RNA targeting of mutations via suppressor tRNAs and deaminases

ABSTRACT

Aspects of the disclosure relate to a gene therapy approach for diseases, disorders, or conditions caused by mutation in the stop codon utilizing modified tRNA. At least 10-15% of all genetic diseases, including muscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, beta thalassemia, Hurler syndrome, and cystic fibrosis, fall into this category. Not to be bound by theory, it is believed that this approach is safer than CRISPR approaches due to minimal off-target effects and the lack of genome level changes.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/490,494, filed Aug. 30, 2019, which application is a U.S. NationalStage Application filed under 35 U.S.C. § 371 and claims priority toInternational Application No. PCT/US2018/020762, filed Mar. 2, 2018,which claims priority under 35 U.S.C. 119(e) to U.S. Ser. No.62/466,961, filed Mar. 3, 2017, and U.S. Ser. No. 62/551,732, filed Aug.29, 2017, the disclosures of each of which are incorporated by referenceherein.

STATEMENT REGARDING GOVERNMENT SUPPORT

This invention was made with government support under Grant No.R01HG009285 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 16, 2020, isnamed Sequence-Listing.txt and is 220,129 bytes in size.

BACKGROUND

Aspects of the disclosure relate to a gene therapy approach fordiseases, disorders, or conditions caused by mutation in the stop codonusing modified tRNA. At least 10-15% of all genetic diseases, includingmuscular dystrophy (e.g. Duchene muscular dystrophy), some cancers, betathalassemia, Hurler syndrome, and cystic fibrosis, fall into thiscategory. Not to be bound by theory, it is believed that this approachis safer than CRISPR or TALEN approaches due to minimal off-targeteffects and the lack of genome level changes.

SUMMARY

Aspects of the disclosure relate to a method for restoring expression ofa protein comprising a point mutation in an RNA sequence encoding theprotein in a subject in need thereof, the method comprising, oralternatively consisting essentially of, or yet further consisting ofadministering to the subject a vector encoding one or more tRNA havingan anticodon sequence that recognizes a codon comprising the pointmutation to the subject, optionally wherein the point mutation resultsin a premature stop codon, optionally wherein the point mutation resultsin a premature stop codon. In some embodiments, the point mutationresults in a nonsense mutation having the DNA sequence TAA and the RNAsequence UAA. In some embodiments, the tRNA is an endogenous tRNA with amodified anticodon stem recognizing the codon comprising the pointmutation. In further embodiments, the tRNA is charged with a serine. Insome embodiments, the tRNA is an orthogonal tRNA charged with anon-canonical amino acid. In further embodiments, the vector furthercomprises a corresponding tRNA synthetase. In some embodiments, thecorresponding synthetase is E. coli Glutaminyl-tRNA synthetase. In someembodiments involving an orthogonal tRNA, the non-canonical amino acidis pyrrolysine. In further embodiments, the pyrrolysine is administeredto the subject by introduction into the diet of the subject. In someembodiments, the vector encodes two tRNA having an anticodon sequencethat recognizes the codon comprising the point mutation. In someembodiments, the protein is dystrophin. In a further aspect, the subjectis a human and is optionally a pediatric patient.

Further method aspects relate to a treating a disease, disorder, orcondition characterized by the presence of a point mutation in an RNAsequence encoding a protein associated with the disease, disorder, orcondition in a subject in need thereof, the method comprising, oralternatively consisting essentially of, or yet further consisting of,administering to the subject a vector encoding one or more tRNA havingan anticodon sequence that recognizes a codon comprising the pointmutation to the subject, optionally wherein the point mutation resultsin a premature stop codon. In some embodiments, the point mutationresults in a nonsense mutation having the DNA sequence TAA and the RNAsequence UAA. In some embodiments, the tRNA is an endogenous tRNA with amodified anticodon stem recognizing the codon comprising the pointmutation. In further embodiments, the tRNA is charged with a serine. Insome embodiments, the tRNA is an orthogonal tRNA charged with anon-canonical amino acid. In further embodiments, the vector furthercomprises a corresponding tRNA synthetase. In some embodiments, thecorresponding synthetase is E. coli Glutaminyl-tRNA synthetase. In someembodiments involving an orthogonal tRNA, the non-canonical amino acidis pyrrolysine. In further embodiments, the pyrrolysine is introduced inthe diet of the subject. In some embodiments, the vector encodes twotRNA having an anticodon sequence that recognizes the codon comprisingthe point mutation. In some embodiments, the disease, disorder, orcondition is selected from the group consisting of the diseases,disorders, and conditions listed in Table 1, optionally characterized bythe presence of a nonsense mutation and/or a premature stop codon. Insome embodiments, the protein is dystrophin. In further embodiments, thedisease, disorder, or condition is muscular dystrophy. In still furtherembodiments, the disease disorder or condition is Duchenne musculardystrophy. In some embodiments, the subject is a human and is optionallya pediatric patient.

Still further aspects disclosed herein relate to a vector encoding oneor more tRNA having an anticodon sequence that recognizes a codoncomprising a point mutation in an RNA sequence encoding a protein,optionally wherein the point mutation results in a premature stop codon.In some embodiments, the point mutation results in a nonsense mutationhaving the DNA sequence TAA and the RNA sequence UAA. In someembodiments, the tRNA is an endogenous tRNA with a modified anticodonstem recognizing the codon comprising the point mutation. In furtherembodiments, the tRNA is charged with a serine. In some embodiments, thetRNA is an orthogonal tRNA charged with a non-canonical amino acid. Infurther embodiments, the vector further comprises a corresponding tRNAsynthetase. In some embodiments, the corresponding synthetase is E. coliGlutaminyl-tRNA synthetase. In some embodiments involving an orthogonaltRNA, the non-canonical amino acid is pyrrolysine. In some embodiments,the vector encodes two tRNA having an anticodon sequence that recognizesthe codon comprising the point mutation. In some embodiments, the vectoris an AAV vector, optionally an AAV8 vector. In some embodiments, theprotein is dystrophin. In a further aspect, the subject is a human andis optionally a pediatric patient.

In another aspect, the disclosure relates to a method for restoringexpression of a protein comprising a point mutation in an RNA sequenceencoding the protein in a subject in need thereof comprisingadministering one or more vectors encoding an ADAR based RNA editingsystem comprising one or more forward guide RNAs for the ADAR (“adRNAs”)and one or more corresponding reverse guide RNAs for the ADAR(“radRNAs”) to the subject, wherein the ADAR based RNA editing systemspecifically edits the point mutation. In some embodiments, the pointmutation results in a nonsense mutation having the DNA sequence TAA andthe RNA sequence UAA. In some embodiments, the ADAR based RNA editingsystem converts UAA to UIA and, optionally, further UIA to UM In someembodiments, the ADAR based RNA editing system converts UAA to UAI. Insome embodiments, optionally those involving nonsense or missensemutations, the RNA targeted in mRNA. In further embodiments, the one ormore vector further encodes a tRNA that targets an amber codon. In someembodiments, the protein is dystrophin. In some embodiments, the pointmutation results in a splice site or missense mutation having the DNAsequence CAG and the RNA sequence CAG. In some embodiments, the ADARbased RNA editing system converts CAG to CIG. In some embodiments,optionally those involving splice site mutations, the RNA targeted ispre-mRNA. In some embodiments, the protein is ornithine transcabamylase.In some embodiments, the ADAR based editing system further comprisesADAR1, ADAR2, the E488Q and E100Q mutants each thereof, a fusion proteincomprising the catalytic domain of an ADAR and a domain which associateswith an RNA hairpin motif, a fusion protein comprising the catalyticdomain of an ADAR and a dead Cas9, or a fusion protein comprising thedouble stranded binding domain of an ADAR and an APOBEC. In furtherembodiments, the domain which associates with an RNA hairpin motif isselected from the group of an MS2 bacteriophage coat protein (MCP) andan N22 peptide. In some embodiments, the method further comprisesadministering an effective amount of an interferon to enhance endogenousADAR1 expression. In still further embodiments, the interferon isinterferon α. In some embodiments, the adRNA comprises one or more RNAhairpin motifs. In some embodiments, the one or more RNA hairpin motifsare selected from the group of an MS2 stem loop and a BoxB loop and/orare stabilized by replacing A-U with G-C. In some embodiments, the adRNAis stabilized through the incorporation of one or more of 2′-O-methyl,2′-O-methyl 3′phosphorothioate, or 2′-O-methyl 3′thioPACE at either orboth termini of the adRNA. In a further aspect, the subject is a humanand is optionally a pediatric patient.

Further method aspects relate to a method of treating a disease,disorder, or condition characterized by the presence of a point mutationin an RNA sequence encoding a protein associated with the disease,disorder, or condition in a subject in need thereof, the methodcomprising, or alternatively consisting essentially of, or yet furtherconsisting of, administering to the subject one or more vectors encodingan ADAR based RNA editing system comprising one or more forward guideRNAs for the ADAR (“adRNAs”) and one or more corresponding reverse guideRNAs for the ADAR (“radRNAs”) to the subject, wherein the ADAR based RNAediting system specifically edits the point mutation. In someembodiments, the point mutation results in a nonsense mutation havingthe DNA sequence TAA and the RNA sequence UAA. In some embodiments, theADAR based RNA editing system converts UAA to UIA and, optionally,further UIA to UII. In some embodiments, the ADAR based RNA editingsystem converts UAA to UAI. In some embodiments, optionally thoseinvolving nonsense or missense mutations, the RNA targeted in mRNA. Infurther embodiments, the one or more vector further encodes a tRNA thattargets an amber codon. In some embodiments, the protein is dystrophin.In some embodiments, the point mutation results in a splice site ormissense mutation having the DNA sequence CAG and the RNA sequence CAG.In some embodiments, the ADAR based RNA editing system converts CAG toCIG. In some embodiments, optionally those involving splice sitemutations, the RNA targeted is pre-mRNA. In some embodiments, theprotein is ornithine transcabamylase. In some embodiments, the ADARbased editing system further comprises ADAR1, ADAR2, the E488Q and E100Qmutants each thereof, a fusion protein comprising the catalytic domainof an ADAR and a domain which associates with an RNA hairpin motif, afusion protein comprising the catalytic domain of an ADAR and a deadCas9, or a fusion protein comprising the double stranded binding domainof an ADAR and an APOBEC. In further embodiments, the domain whichassociates with an RNA hairpin motif is selected from the group of anMS2 bacteriophage coat protein (MCP) and an N22 peptide. In someembodiments, the method further comprises administering an effectiveamount of an interferon to enhance endogenous ADAR1 expression. In stillfurther embodiments, the interferon is interferon α. In someembodiments, the adRNA comprises one or more RNA hairpin motifs. In someembodiments, the one or more RNA hairpin motifs are selected from thegroup of an MS2 stem loop and a BoxB loop and/or are stabilized byreplacing A-U with G-C. In some embodiments, the adRNA is stabilizedthrough the incorporation of one or more of 2′-O-methyl, 2′-O-methyl3′phosphorothioate, or 2′-O-methyl 3′thioPACE at either or both terminiof the adRNA. In some embodiments, the disease, disorder, or conditionis selected from the group consisting of the diseases, disorders, andconditions listed in Table 1. In further embodiments, the protein isdystrophin and the disease, disorder, or condition is musculardystrophy. In still further embodiments, the disease disorder orcondition is Duchenne muscular dystrophy. In some embodiments, thesubject is a human and is optionally a pediatric patient.

Additional aspects relate to a recombinant expression system comprisingone or more vectors encoding an ADAR based RNA editing system comprisingone or more forward guide RNAs for the ADAR (“adRNAs”) and one or morecorresponding reverse guide RNAs for the ADAR (“radRNAs”) to thesubject, wherein the ADAR based RNA editing system specifically edits apoint mutation in an RNA sequence encoding a protein. In someembodiments, the point mutation results in a nonsense mutation havingthe DNA sequence TAA and the RNA sequence UAA. In some embodiments, theADAR based RNA editing system converts UAA to UIA and, optionally,further UIA to UII. In some embodiments, the ADAR based RNA editingsystem converts UAA to UAI. In some embodiments, optionally thoseinvolving nonsense or missense mutations, the RNA targeted in mRNA. Infurther embodiments, the one or more vector further encodes a tRNA thattargets an amber codon. In some embodiments, the protein is dystrophin.In some embodiments, the point mutation results in a splice site ormissense mutation having the DNA sequence CAG and the RNA sequence CAG.In some embodiments, the ADAR based RNA editing system converts CAG toCIG. In some embodiments, optionally those involving splice sitemutations, the RNA targeted is pre-mRNA. In some embodiments, theprotein is ornithine transcabamylase. In some embodiments, the ADARbased editing system further comprises ADAR1, ADAR2, the E488Q and E100Qmutants each thereof, a fusion protein comprising the catalytic domainof an ADAR and a domain which associates with an RNA hairpin motif, afusion protein comprising the catalytic domain of an ADAR and a deadCas9, or a fusion protein comprising the double stranded binding domainof an ADAR and an APOBEC. In further embodiments, the domain whichassociates with an RNA hairpin motif is selected from the group of anMS2 bacteriophage coat protein (MCP) and an N22 peptide. In someembodiments, the adRNA comprises one or more RNA hairpin motifs. In someembodiments, the one or more RNA hairpin motifs are selected from thegroup of an MS2 stem loop and a BoxB loop and/or are stabilized byreplacing A-U with G-C. In some embodiments, the adRNA is stabilizedthrough the incorporation of one or more of 2′-O-methyl, 2′-O-methyl3′phosphorothioate, or 2′-O-methyl 3′thioPACE at either or both terminiof the adRNA. In a further aspect, the subject is a human and isoptionally a pediatric patient.

Still further aspects relate to a composition comprising any one or moreof the vectors disclosed herein and optionally one or more carriers,such as a pharmaceutically acceptable carrier. In some embodiments, thecomposition further comprises an effective amount of an interferon toenhance endogenous ADAR1 expression. In still further embodiments, theinterferon is interferon α.

Some aspects disclosed herein relate to methods for restoring expressionof a protein in a subject in need thereof, the method comprising, oralternatively consisting essentially of, or yet further consisting of,administering to the subject a tRNA having an anticodon sequence thatrecognizes a mutation in an RNA sequence encoding the protein or avector encoding one or more of said tRNA to the subject. In someembodiments, the mutation is a nonsense mutation, optionally a prematurestop codon. In some embodiments, the nonsense mutation is TAA in DNA andUAA in RNA. In some embodiments, the tRNA is a modified endogenous tRNAcharged with a canonical amino acid. In some embodiments, the canonicalamino acid is serine. In some embodiments, the tRNA is an orthogonaltRNA charged with a non-canonical amino acid. In some embodiments, theorthogonal tRNA has a corresponding synthetase. In some embodiments, thecorresponding synthetase is E. coli Glutaminyl-tRNA synthetase. In someembodiments, the non-canonical amino acid is introduced or administeredto the subject (e.g. through food), allowing for the induction of theorthogonal tRNA activity. In some embodiments, the non-canonical aminoacid is pyrrolysine. In some embodiments, the tRNA targets an ambercodon. In some embodiments, the tRNA targets an ochre codon. In someembodiments, the tRNA targets an opal codon. In some embodiments, theprotein is dystrophin. In a further aspect, the subject is a human andis optionally a pediatric patient.

Further aspects disclosed herein relate to methods of a disease,disorder, or condition characterized by a protein deficiency in asubject in need thereof, the method comprising, or alternativelyconsisting essentially or, or yet further consisting of administering atRNA having an anticodon sequence that recognizes a mutation in an RNAsequence encoding the protein or a vector encoding one or more of saidtRNA to the subject. In some embodiments, the mutation is a nonsensemutation, optionally a premature stop codon. In some embodiments, thenonsense mutation is TAA in DNA and UAA in RNA. In some embodiments, thetRNA is a modified endogenous tRNA charged with a canonical amino acid.In some embodiments, the canonical amino acid is serine. In someembodiments, the tRNA is an orthogonal tRNA charged with a non-canonicalamino acid. In some embodiments, the orthogonal tRNA has a correspondingsynthetase. In some embodiments, the corresponding synthetase is E. coliGlutaminyl-tRNA synthetase. In some embodiments, the non-canonical aminoacid is administered or introduced to the subject (e.g. through food),allowing for the induction of the orthogonal tRNA activity. In someembodiments, the non-canonical amino acid is pyrrolysine. In someembodiments, the tRNA targets an amber codon. In some embodiments, thetRNA targets an ochre codon. In some embodiments, the tRNA targets anopal codon. In some embodiments, the protein deficiency is a dystrophindeficiency. In some embodiments, the disease, disorder, or condition ismuscular dystrophy. In some embodiments, the muscular dystrophy isDuchene muscular dystrophy. In a further aspect, the subject is a humanand is optionally a pediatric patient.

Other aspects relate to a vector encoding one or more tRNA having ananticodon sequence that recognizes a mutation in an RNA sequenceencoding the protein. In some embodiments, the mutation is a nonsensemutation, optionally a premature stop codon. In some embodiments, thenonsense mutation is TAA in DNA and UAA in RNA. In some embodiments, thetRNA is a modified endogenous tRNA charged with a canonical amino acid.In some embodiments, the canonical amino acid is serine. In someembodiments, the tRNA is an orthogonal tRNA charged with a non-canonicalamino acid. In some embodiments, the orthogonal tRNA has a correspondingsynthetase. In some embodiments, the corresponding synthetase is E. coliGlutaminyl-tRNA synthetase. In some embodiments, the vector furthercomprises the corresponding synthetase. In some embodiments, thenon-canonical amino acid is introduced or administered to the subject(e.g. through food), allowing for the induction of the orthogonal tRNAactivity. In some embodiments, the non-canonical amino acid ispyrrolysine. In some embodiments, the tRNA targets an amber codon. Insome embodiments, the tRNA targets an ochre codon. In some embodiments,the tRNA targets an opal codon. In some embodiments, the protein isdystrophin. In some embodiments, the mutation is a nonsense mutation,optionally a premature stop codon. In some embodiments, the vector is anAdeno-Associated Virus (AAV) vector. In some embodiments, the AAV vectoris an AAV8 vector.

Additional aspects of this disclosure relate to on-demand, in vivoproduction of therapeutic proteins, such as, but not limited to, (i)insulin; (ii) neutralizing antibodies for viruses (e.g. HIV, HCV, HPV,influenza) and bacteria (e.g. Staph Aureus; drug resistant strains).Such method aspects comprise administering to a subject a vectorencoding the therapeutic protein with a mutation in its sequence and atRNA having an anticodon sequence that recognizes the mutation in theRNA sequence encoding the therapeutic protein or a vector encoding oneor more of said tRNA. Accordingly, any of the methods and vectorsdisclosed hereinabove relating to a tRNA having an anticodon sequencethat recognizes a mutation in an RNA sequence encoding the protein or avector encoding one or more of said tRNA may be applied to this aspect,as well.

Some aspects disclosed herein relate to methods for restoring expressionof a protein in a subject in need thereof comprising administering anADAR2 based RNA editing system comprising an ADAR2, one or more forwardguide RNAs for the ADAR2 (“adRNAs”), and one or more correspondingreverse guide RNAs for the ADAR2 (“radRNAs”) to the subject, wherein theADAR2 based RNA editing system specifically edits a mutation in an RNAsequence encoding the protein or one or more vectors encoding saidADAR2, adRNAs, radRNAs. In some embodiments, the ADAR2 based RNA editingsystem changes adenosine (A) to inosine (I), which is read duringtranslation as guanosine (G). In some embodiments, the mutation is anonsense mutation. In some embodiments, the nonsense mutation is TAA inDNA and UAA in RNA. In some embodiments, the ADAR2 based RNA editingsystem causes point mutations at one or more adenosines (A) in thenonsense mutation. In some embodiments, the ADAR2 based RNA editingsystem converts UAA to UIA (read as UGA). In further embodiments, theADAR2 based RNA editing system converts UIA (read as UGA) to UII (readas UGG). In some embodiments, the ADAR2 based RNA editing systemconverts UAA to UAI (read as UAG). In some embodiments, the methodfurther comprises administering a tRNA, such as one disclosedhereinabove, that recognizes the codon encoded by the ADAR2 editedsequence. In some embodiments, the tRNA is a modified endogenous tRNAcharged with a canonical amino acid. In some embodiments, the canonicalamino acid is serine. In some embodiments, the tRNA is an orthogonaltRNA charged with a non-canonical amino acid. In some embodiments, theorthogonal tRNA has a corresponding synthetase. In some embodiments, thecorresponding synthetase is E. coli Glutaminyl-tRNA synthetase. In someembodiments, the non-canonical amino acid is introduced to the subject(e.g. through food), allowing for the induction of the orthogonal tRNAactivity. In some embodiments, the non-canonical amino acid ispyrrolysine. In some embodiments, the tRNA targets an amber codon. Insome embodiments, the tRNA targets an ochre codon. In some embodiments,the tRNA targets an opal codon. In some embodiments, the proteindeficiency is a dystrophin deficiency. In some embodiments, the disease,disorder, or condition is muscular dystrophy. In some embodiments, themuscular dystrophy is Duchene muscular dystrophy.

Further aspects disclosed herein relate to methods of a disease,disorder, or condition characterized by a protein deficiency in asubject in need thereof comprising administering an ADAR2 based RNAediting system comprising an ADAR2, one or more forward guide RNAs forthe ADAR2 (“adRNAs”), and one or more corresponding reverse guide RNAsfor the ADAR2 (“radRNAs”) to the subject, wherein the ADAR2 based RNAediting system specifically edits a mutation in an RNA sequence encodingthe protein or one or more vectors encoding said ADAR2, adRNAs, radRNAs.In some embodiments, the ADAR2 based RNA editing system changesadenosine (A) to inosine (I), which is read during translation asguanosine (G). In some embodiments, the mutation is a nonsense mutation.In some embodiments, the nonsense mutation is TAA. In some embodiments,the ADAR2 based RNA editing system causes point mutations at one or moreadenosines (A) in the nonsense mutation. In some embodiments, the ADAR2based RNA editing system converts UAA to UIA (read as UGA). In furtherembodiments, the ADAR2 based RNA editing system converts UIA (read asUGA) to UII (read as UGG). In some embodiments, the ADAR2 based RNAediting system converts UAA to UAI (read as UAG). In some embodiments,the method further comprises administering a tRNA, such as one disclosedhereinabove, that recognizes the codon encoded by the ADAR2 editedsequence. In some embodiments, the tRNA is a modified endogenous tRNAcharged with a canonical amino acid. In some embodiments, the canonicalamino acid is serine. In some embodiments, the tRNA is an orthogonaltRNA charged with a non-canonical amino acid. In some embodiments, theorthogonal tRNA has a corresponding synthetase. In some embodiments, thecorresponding synthetase is E. coli Glutaminyl-tRNA synthetase. In someembodiments, the non-canonical amino acid is introduced to the subject(e.g. through food), allowing for the induction of the orthogonal tRNAactivity. In some embodiments, the non-canonical amino acid ispyrrolysine. In some embodiments, the tRNA targets an amber codon. Insome embodiments, the tRNA targets an ochre codon. In some embodiments,the tRNA targets an opal codon. In some embodiments, the proteindeficiency is a dystrophin deficiency. In some embodiments, the disease,disorder, or condition is muscular dystrophy. In some embodiments, themuscular dystrophy is Duchene muscular dystrophy.

Other aspects relate to a recombinant expression system comprising oneor more vectors encoding an ADAR2 based RNA editing system comprisingone or more of an ADAR2, one or more forward guide RNAs for the ADAR2(“adRNAs”), and one or more corresponding reverse guide RNAs for theADAR2 (“radRNAs”), wherein the ADAR2 based RNA editing systemspecifically edits a mutation in an RNA sequence encoding a protein. Insome embodiments, the ADAR2 changes adenosine (A) to inosine (I), whichis read during translation as guanosine (G). In some embodiments, oneadRNA/radRNA pair guides the conversion of UAA to UIA (read as UGA). Infurther embodiments, a second adRNA/radRNA pair guides the conversion ofUIA (read as UGA) to UII (read as UGG). In some embodiments, oneadRNA/radRNA pair guides the conversion of UAA to UAI (read as UAG). Insome embodiments, the one or more vectors or an additional vectorfurther encodes a tRNA, such as one disclosed hereinabove, thatrecognizes the codon encoded by the ADAR2 edited sequence. In someembodiments, the tRNA is a modified endogenous tRNA charged with acanonical amino acid. In some embodiments, the canonical amino acid isserine. In some embodiments, the tRNA is an orthogonal tRNA charged witha non-canonical amino acid. In some embodiments, the orthogonal tRNA hasa corresponding synthetase. In some embodiments, the correspondingsynthetase is E. coli Glutaminyl-tRNA synthetase. In some embodiments,the non-canonical amino acid is introduced to the subject (e.g. throughfood), allowing for the induction of the orthogonal tRNA activity. Insome embodiments, the non-canonical amino acid is pyrrolysine. In someembodiments, the tRNA targets an amber codon. In some embodiments, thetRNA targets an ochre codon. In some embodiments, the tRNA targets anopal codon. In some embodiments, the protein is dystrophin. In someembodiments, the mutation is a nonsense mutation. In some embodiments,the vector is an Adeno-Associated Virus (AAV) vector. In someembodiments, the AAV vector is an AAV8 vector.

Additional aspects of this disclosure relate to on-demand, in vivoproduction of therapeutic proteins, such as, but not limited to, (i)insulin; (ii) neutralizing antibodies for viruses (e.g. HIV, HCV, HPV,influenza) and bacteria (e.g. Staph Aureus; drug resistant strains).Such method aspects comprise administering to a subject a vectorencoding the therapeutic protein with a mutation in its sequence and anADAR2 based RNA editing system comprising an ADAR2, one or more forwardguide RNAs for the ADAR2 (“adRNAs”), and one or more correspondingreverse guide RNAs for the ADAR2 (“radRNAs”), wherein the ADAR2 basedRNA editing system specifically edits a mutation in an RNA sequenceencoding the protein or one or more vectors encoding said ADAR2, adRNAs,radRNAs. Accordingly, any of the methods and vectors disclosedhereinabove relating to an ADAR2 based RNA editing system specificallyedits a mutation in an RNA sequence encoding the protein or a vectorencoding one or more vectors encoding said ADAR2, adRNAs, radRNAs.

PARTIAL SEQUENCE LISTING

mU6, tRNA(U25C) Amber (SEQ ID NO: 1)tcccggggtttccgccaTTTTTTGGTACTGAGtCGCCCaGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGggaaacctgatcatgtagatcgaaCggactCTAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTCCTAGACCCAGCTTTCTTGTACAAAGTTGG mU6, tRNA(U25C) Ochre(SEQ ID NO: 2)tcccggggtttccgccaTTTTTTGGTACTGAGtCGCCCaGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGggaaacctgatcatgtagatcgaaCggactTTAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTCCTAGACCCAGCTTTCTTGTACAAAGTTGG mU6, tRNA(U25C) Opal(SEQ ID NO: 3)tcccggggtttccgccaTTTTTTGGTACTGAGtCGCCCaGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGggaaacctgatcatgtagatcgaaCggactTCAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTCCTAGACCCAGCTTTCTTGTACAAAGTTGG MmPylRS (AfIII)(SEQ ID NO: 4)CAGCCTCCGGACTCTAGAGGATCGAACCCTTAAGgccaccATGGATAAGAAACCTTTGAACACTCTCATTAGTGCGACAGGGCTCTGGATGTCCCGAACGGGGACTATACACAAGATAAAACACCATGAGGTCTCAAGGAGCAAAATCTATATCGAGATGGCATGCGGCGACCATCTTGTGGTAAATAATAGTAGGTCCTCCAGGACGGCAAGAGCACTCCGACATCACAAGTACAGAAAAACCTGCAAACGGTGTAGGGTATCCGACGAAGACTTGAACAAATTTTTGACTAAGGCCAACGAGGATCAAACTTCTGTCAAAGTGAAAGTGGTTTCTGCTCCTACCCGAACTAAGAAGGCCATGCCCAAGTCCGTGGCAAGGGCACCCAAGCCACTCGAAAATACTGAGGCCGCTCAGGCCCAACCATCCGGTAGTAAGTTCAGTCCAGCCATACCCGTAAGTACCCAAGAATCTGTCAGTGTGCCGGCCTCAGTTTCCACATCTATAAGTTCAATTTCTACAGGAGCGACGGCCTCCGCCCTCGTCAAGGGTAACACAAACCCGATAACTTCTATGAGTGCCCCCGTACAGGCATCCGCACCAGCACTGACGAAGTCTCAAACTGATAGGCTGGAAGTGCTCTTGAATCCGAAGGACGAGATATCTCTTAACTCCGGTAAACCTTTCCGGGAGCTGGAAAGTGAACTTCTCAGCCGGCGAAAAAAAGACCTCCAGCAAATTTACGCAGAGGAAAGGGAGAACTATCTGGGGAAGTTGGAACGAGAGATCACCCGATTCTTTGTCGATCGCGGATTTTTGGAGATTAAAAGCCCAATTCTCATCCCCCTTGAATATATCGAACGAATGGGAATCGACAATGATACGGAGTTGTCCAAGCAGATTTTCCGCGTAGACAAGAACTTTTGTCTTCGACCCATGCTCGCTCCGAACCTCTACAATTACTTGAGAAAGTTGGACAGAGCGCTCCCGGACCCGATCAAGATATTTGAGATCGGTCCTTGTTATAGAAAGGAGAGTGATGGAAAAGAACACCTCGAAGAGTTCACGATGCTGAACTTCTGCCAAATGGGTTCTGGCTGCACACGGGAGAATCTCGAAAGCATCATTACAGATTTCCTTAACCATCTGGGGATAGACTTTAAAATAGTGGGTGACAGCTGTATGGTATACGGAGATACCTTGGACGTAATGCACGGGGATCTTGAGCTTTCCTCCGCCGTGGTTGGACCTATACCGTTGGACCGGGAGTGGGGAATCGACAAACCGTGGATAGGCGCCGGTTTCGGCCTTGAAAGACTCCTCAAAGTCAAGCATGATTTCAAAAACATAAAACGGGCTGCTCGCTCCGAATCTTATTACAACGGTATAAGTACGAACCTGTGATAATAGCTTAAGGGTTCGATCCCTACtGGTTAGTAATGAGTTTA tRNAs Amber suppression: (SEQ ID NO: 5)ggaaacctgatcatgtagatcgaatggactctaaatccgttcagccgggttagattcccggggtttccgccaAmber suppression (2): (SEQ ID NO: 6)ggggggtggatcgaatagatcacacggactctaaattcgtgcaggcgggtgaaactcccgtactccccgccaOchre suppression (SEQ ID NO: 7)ggaaacctgatcatgtagatcgaatggactttaaatccgttcagccgggttagattcccggggtttccgccaOpal suppression: (SEQ ID NO: 8)ggaaacctgatcatgtagatcgaatggacttcaaatccgttcagccgggttagattcccggggtttccgccaSynthetase: (SEQ ID NO: 9)ATGGATAAAAAACCATTAGATGTTTTAATATCTGCGACCGGGCTCTGGATGTCCAGGACTGGCACGCTCCACAAAATCAAGCACCATGAGGTCTCAAGAAGTAAAATATACATTGAAATGGCGTGTGGAGACCATCTTGTTGTGAATAATTCCAGGAGTTGTAGAACAGCCAGAGCATTCAGACATCATAAGTACAGAAAAACCTGCAAACGATGTAGGGTTTCGGACGAGGATATCAATAATTTTCTCACAAGATCAACCGAAAGCAAAAACAGTGTGAAAGTTAGGGTAGTTTCTGCTCCAAAGGTCAAAAAAGCTATGCCGAAATCAGTTTCAAGGGCTCCGAAGCCTCTGGAAAATTCTGTTTCTGCAAAGGCATCAACGAACACATCCAGATCTGTACCTTCGCCTGCAAAATCAACTCCAAATTCGTCTGTTCCCGCATCGGCTCCTGCTCCTTCACTTACAAGAAGCCAGCTTGATAGGGTTGAGGCTCTCTTAAGTCCAGAGGATAAAATTTCTCTAAATATGGCAAAGCCTTTCAGGGAACTTGAGCCTGAACTTGTGACAAGAAGAAAAAACGATTTTCAGCGGCTCTATACCAATGATAGAGAAGACTACCTCGGTAAACTCGAACGTGATATTACGAAATTTTTCGTAGACCGGGGTTTTCTGGAGATAAAGTCTCCTATCCTTATTCCGGCGGAATACGTGGAGAGAATGGGTATTAATAATGATACTGAACTTTCAAAACAGATCTTCCGGGTGGATAAAAATCTCTGCTTGAGGCCAATGCTTGCCCCGACTCTTTACAACTATCTGCGAAAACTCGATAGGATTTTACCAGGCCCAATAAAAATTTTCGAAGTCGGACCTTGTTACCGGAAAGAGTCTGACGGCAAAGAGCACCTGGAAGAATTTACTATGGTGAACTTCTGTCAGATGGGTTCGGGATGTACTCGGGAAAATCTTGAAGCTCTCATCAAAGAGTTTCTGGACTATCTGGAAATCGACTTCGAAATCGTAGGAGATTCCTGTATGGTCTTTGGGGATACTCTTGATATAATGCACGGGGACCTGGAGCTTTCTTCGGCAGTCGTCGGGCCAGTTTCTCTTGATAGAGAATGGGGTATTGACAAACCATGGATAGGTGCAGGTTTTGGTCTTGAACGCTTGCTCAAGGTTATGCACGGCTTTAAAAACATTAAGAGGGCATCAAGGTCCGAATCTTACTATAATGGGATTTCAACCAATCTGTAAEGFP: (SEQ ID NO: 10)atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa EGFP Amber: (SEQ ID NO: 11)Atggtgagcaagggcgaggagagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctagggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctagtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaatga EGFP Ochre: (SEQ ID NO: 12)atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctaaggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtagaagtaatga EGFP Opal: (SEQ ID NO: 13)Atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctgaggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaatga MbPylRS (SEQ ID NO: 14)        10         20         30         40         50MDKKPLDVLI SATGLWMSRT GTLHKIKHHE VSRSKIYIEM ACGDHLVVNN        60         70         80         90        100SRSCRTARAF RHHKYRKTCK RCRVSDEDIN NFLTRSTESK NSVKVRVVSA       110        120        130        140        150PKVKKAMPKS VSRAPKPLEN SVSAKASTNT SRSVPSPAKS TPNSSVPASA       160        170        180        190        200PAPSLTRSQL DRVEALLSPE DKISLNMAKP FRELEPELVT RRKNDFQRLY       210        220        230        240        250TNDREDYLGK LERDITKFFV DRGFLEIKSP ILIPAEYVER MGINNDTELS       260        270        280        290        300KQIFRVDKNL CLRPMLAPTL YNYLRKLDRI LPGPIKIFEV GPCYRKESDG       310        320        330        340        350KEHLEEFTMV NECQMGSGCT RENLEALIKE FLDYLEIDFE IVGDSCMVYG       360        370        380        390        400DTLDIMHGDL ELSSAVVGPV SLDREWGIDK PWIGAGFGLE RLLKVMHGFK        410NIKRASRSES YYNGISTNL MmPylRS (uniprot) (SEQ ID NO: 15)        10         20         30         40         50MDKKPLNTLI SATGLWMSRT GTIHKIKHHE VSRSKIYIEM ACGDHLVVNN        60         70         80         90        100SRSSRTARAL RHHKYRKTCK RCRVSDEDLN KFLTKANEDQ TSVKVKVVSA       110        120        130        140        150PTRTKKAMPK SVARAPKPLE NTEAAQAQPS GSKFSPAIPV STQESVSVPA       160        170        180        190        200SVSTSISSIS TGATASALVK GNTNPITSMS APVQASAPAL TKSQTDRLEV       210        220        230        240        250LLNPKDEISL NSGKPFRELE SELLSRRKKD LQQIYAEERE NYLGKLEREI       260        270        280        290        300TRFFVDRGFL EIKSPILIPL EYIERMGIDN DTELSKQIFR VDKNFCLRPM       310        320        330        340        350LAPNLYNYLR KLDRALPDPI KIFEIGPCYR KESDGKEHLE EFTMLNFCQM       360        370        380        390        400GSGCTRENLE SIITDFLNHL GIDFKIVGDS CMVYGDTLDV MHGDLELSSA       410        420        430        440        450VVGPIPLDRE WGIDKPWIGA GFGLERLLKV KHDFKNIKRA ARSESYYNGI STNLPylT* (Amber) (SEQ ID NO: 16)ggaaacctgatcatgtagatcgaaCggactCTAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTPylT* (Ochre) (SEQ ID NO: 17)ggaaacctgatcatgtagatcgaaCggactTTAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTPylT* (Opal) (SEQ ID NO: 18)ggaaacctgatcatgtagatcgaaCggactTCAaatccgttcagccgggttagattcccggggtttccgccaTTTTTTMouse U6 primers (SEQ ID NO: 19)tcccggggtttccgccaTTTTTTGGTACTGAGtCGCCCaGTCTCAGAT (SEQ ID NO: 20)CAAACAAGGCTTTTCTCCAAGGGATAT tRNA (U25C) Amber_F: (SEQ ID NO: 21)CCTTGGAGAAAAGCCTTGTTTGggaaacctgatcatgtagatcgaarggactCTAaatccgttcagccgggCommon reverse: PylT (SEQ ID NO: 22)ggaaacctgatcatgtagatcgaatggactCTAaatccgttcagccgggttagattcccggggtttccgccaPylT*(U25C) (SEQ ID NO: 23)ggaaacctgatcatgtagatcgaaCggactCTAaatccgttcagccgggttagattcccggggtttccgcca1. Arg tRNA (opal) (E-Cadherin paper) (SEQ ID NO: 24)GGCCGCGTGGCCTAATGGATAAGGCGTCTGACT

GATCAGAAGATTGCAGGTTCGAGTCCTGCCGCGGTCG2. Arg tRNA (opal) (Xeroderma paper) (SEQ ID NO: 25)GACCACGTGGCCTAATGGATAAGGCGTCTGACT

GATCAGAAGATTGAGGGTTCGAATCCCTTCGTGGTTA 3. Serine tRNA (amber)(SEQ ID NO: 26) GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACT

AATCCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGACTACG 4. Leucine tRNA (amber)(SEQ ID NO: 27) GTCAGGATGGCCGAGTGGTCTAAGGCGCCAGACT

GTTCTGGTCTCCAATGGAGGCGTGGGTTCGAATCCCACTTCTGACA Forward: (SEQ ID NO: 28)TTGTGGAAAGGACGAAACACC Reverse: (SEQ ID NO: 29)ACAAGAAAGCTGGGTCTAGGCTAGCAAAAAAtRNA_Leu_Am_F (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 30) TTGTGGAAAGGACGAAACACCGGTCAGGATGGCCGAGTGGTCTAAGGCGCCAGACT

GTTCTGGTCTCCAATGGtRNA_Leu_Oc_F (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 31) TTGTGGAAAGGACGAAACACCGGTCAGGATGGCCGAGTGGTCTAAGGCGCCAGACT

GTTCTGGTCTCCAATGGtRNA_Leu_Op_F (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 32) TTGTGGAAAGGACGAAACACCGGTCAGGATGGCCGAGTGGTCTAAGGCGCCAGACT

GTTCTGGTCTCCAATGGtRNA_Leu_R (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 33)ACAAGAAAGCTGGGTCTAGGCTAGCAAAAAATGTCAGAAGTGGGATTCGAACCCACGCCTCCATTGGAGACCAGAACtRNA_Ser_Am_F (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 34) TTGTGGAAAGGACGAAACACCGGTAGTCGTGGCCGAGTGGTTAAGGCGATGGACT

AATCCATTGGGGTTTCCtRNA_Ser_Oc_F (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 35) TTGTGGAAAGGACGAAACACCGGTAGTCGTGGCCGAGTGGTTAAGGCGATGGACT

AATCCATTGGGGTTTCCtRNA_Ser_Op_(overlaps with vector, bold; anti-codon sequences, bold underline)F:(SEQ ID NO: 36) TTGTGGAAAGGACGAAACACCGGTAGTCGTGGCCGAGTGGTTAAGGCGATGGACT

AATCCATTGGGGTTTCCtRNA_Ser_R (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 37)ACAAGAAAGCTGGGTCTAGGCTAGCAAAAAACGTAGTCGGCAGGATTCGAACCTGCGCGGGGAAACCCCAATGGATTtRNA_Arg_Am_F (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 38) TTGTGGAAAGGACGAAACACCGGACCACGTGGCCTAATGGATAAGGCGTCTGACT

GATCAGAAGATTGAGGGTTtRNA_Arg_Oc_F (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 39) TTGTGGAAAGGACGAAACACCGGACCACGTGGCCTAATGGATAAGGCGTCTGACT

GATCAGAAGATTGAGGGTTtRNA_Arg_Op_F (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 40) TTGTGGAAAGGACGAAACACCGGACCACGTGGCCTAATGGATAAGGCGTCTGACT

GATCAGAAGATTGAGGGTTtRNA_Arg_R (overlaps with vector, bold; anti-codon sequences, bold underline):(SEQ ID NO: 41)ACAAGAAAGCTGGGTCTAGGCTAGCAAAAAATAACCACGAAGGGATTCGAACCCTCAATCTTCTGATCmU6_tRNA_ser_oc: (SEQ ID NO: 42)GTACTGAGtCGCCCaGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTGGTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTTTAAATCCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGACTACGTTTTTTmU6_tRNA_ser_oc_Nhe1_insert_F: (SEQ ID NO: 43)AATCCTGCCGACTACGTTTTTTGTACTGAGtCGCCCAGTCTadRNA (premature stop codon target, bold; edited bases, bold underline):Sequential edits: (SEQ ID NO: 44) TTTGAAAGAGCAATA

AAT (SEQ ID NO: 45) CTTTGAAAGAGCAAT

GAA Dual edits: (SEQ ID NO: 46) TTTGAAAGAGCAAT

AATradRNA (premature stop codon target, bold; edited bases, bold underline):Sequential edits: (SEQ ID NO: 47) Ata

AATGGCTTCAACTAT (SEQ ID NO: 48) AAt

gAATGGCTTCAACTA Dual edits: (SEQ ID NO: 49) AAt

AATGGCTTCAACTA OTC target (edited bases, bold): (SEQ ID NO: 50)TCACAGACACCGCTCAGTTTGTOptimization of the length of adRNA and distance of the edit from the ADAR2 recruitingdomain (Length of adRNA—distance of edit from ADAR2 recruiting domain):16-5: (SEQ ID NO: 51) atgccaccTGGggcaa 16-6: (SEQ ID NO: 52)tgccaccTGGggcaag 16-7: (SEQ ID NO: 53) gccaccTGGggcaagc 18-6:(SEQ ID NO: 54) gatgccaccTGGggcaag 20-6: (SEQ ID NO: 55)gcgatgccaccTGGggcaag ADAR2 recruiting region v1: (SEQ ID NO: 56)GGGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACCTADAR2 recruiting region v2: (SEQ ID NO: 57)GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC Hairpin (3′) (FIG. 8):(SEQ ID NO: 58) GGGCCCTCTTCAGGGCCCTCTAGA Hairpin (3′) (FIG. 10):(SEQ ID NO: 59) atcgccctgaaaag Toe hold (5′): (SEQ ID NO: 60)gccaccTGGgg

List of Suppressor tRNA Sequences:

Sup- pressor tRNAs Sequence (5′ to 3′) SerineGTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTNNNAATCCATTGGGGTTTCCCCGCGCAGGTTCGAATCCTGCCGACTACG  (SEQ ID NO: 61) LeucineGTCAGGATGGCCGAGTGGTCTAAGGCGCCAGACTTNNNGTTVTGGTCTCCAATGGAGGCGTGGGTTCGAATCCCACTTCTG  ACA (SEQ ID NO: 62) Arg-GACCACGTGGCCTAATGGATAAGGCGTCTGACTNNNGATC inineAGAAGATTGAGGGTTCGAATCCCTTCGTGGTTA  (SEQ ID NO: 63)

NNN—Anticodon

In endogenous tRNA, the tRNA is modified to recognize the codoncomprising the point mutation by including the complementary sequence atthe NNN position noted herein above. As clarified in more detail below,the NNN sequences in amber, ochre, and opal tRNA are as follows: Amber:NNN═CTA; Ochre: NNN=TCA; Opal: NNN=TTA.

List of primers for next generation sequencing (NGS) analyses.

Name Sequence (5′ to 3′) NGS_DMD_F1 GTGTTACTGAATATGAAATAATGGAGGA (SEQ ID NO: 64) NGS_DMD_R1 ATTTCTGGCATATTTCTGAAGGTG  (SEQ ID NO: 65)NGS_DMD_F2 CTCTCTGTACCTTATCTTAGTGTTACTGA  (SEQ ID NO: 66) NGS_DMD_R2CTCTTCAAATTCTGACAGATATTTCTGGC  (SEQ ID NO: 67) NGS_OTC_FACCCTTCCTTTCTTACCACACA  (SEQ ID NO: 68) NGS_OTC_RCAGGGTGTCCAGATCTGATTGTT  (SEQ ID NO: 69) NGS_OTC_R2CTTCTCTTTTAAACTAACCCATCAGAGTT  (SEQ ID NO: 70)

List of adRNA antisense sequences and corresponding ADAR2 recruitingscaffold used for in vivo RNA editing studies. In some embodiments, therecruiting scaffold v2—disclosed in prior paragraph, is used with thesesequences.

Name adRNA antisense sequence (3′ to 5′) OTCTGTCTGTGGCGAGCCAAACA (SEQ ID NO: 71) DMDACTTTCTCGTTACCTTACCG (SEQ ID NO: 72)

MCP-Linker-ADAR1-NLS (optional sequence in brackets)MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGS KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPVGSGSGSGPKKRKV[AA]* (SEQ ID NO: 73)MCP-Linker-ADAR2 (optional sequence in brackets)MGPKKKRKVAAGSGSGSMASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY GGSGGSGGS MLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT[P]* (SEQ ID NO: 74)N22p-Linker-ADAR1-NLS (optional sequence in brackets)MGNARTRRRERRAEKQAQWKAANGGGGTSGSGSGS PAGGGAPGSGGGS KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPVGSGSGSGPKKRKV[AA]* (SEQ ID NO: 75)Nuclear Localization Sequence-Linker-N22p-Linker-ADAR2 (optionalsequence in brackets) [MG]PKKKRKVAAGSGSGSMGNARTRRRERRAEKQAQWKAANGGGGTSGSGSG S PAGGGAPGSGGGSMLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT[P]* (SEQ ID NO: 76)MCP-Linker-ADAR1 (E1008Q)-NLS (optional sequence in brackets)MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGS KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGQGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPVGSGSGSGPKKRKV[AA]* (SEQ ID NO: 77)Nuclear Localization Sequence-Linker-MCP-Linker-ADAR2 (E4880)(optional sequence in brackets) [MG]PKKKRKVAAGSGSGSMASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY GGSGGSGGS MLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGQGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT[P]* (SEQ ID NO: 78)N22p-Linker-ADAR1 (E1008Q) (optional sequence in brackets)MGNARTRRRERRAEKQAQWKAANGGGGTSGSGSGS PAGGGAPGSGGGS KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGQGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPVGSGSGSGPKKRKV[AA]* (SEQ ID NO: 79)Nuclear Localization Sequence-Linker-N22p-Linker-ADAR2 (E488Q)[MG]PKKKRKVAAGSGSGS MGNARTRRRERRAEKQAQWKAANGGGGTSGSGSG S PAGGGAPGSGGGSMLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGQGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT[P]* (SEQ ID NO: 80)Nuclear Localization Sequence-Linker-MCP-Linker-hAPOPEC1[MG]PKKKRKVAAGSGSGS MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY GGSGGSGGS MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR* (SEQ ID NO: 81)Nuclear Localization Sequence-Linker-MCP-Linker-rAPOBEC1[MG]PKKKRKVAAGSGSGS MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY GGSGGSGGS MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK* (SEQ ID NO: 82)dsRBD-Linker-rAPOBEC1MDIEDEENMSSSSTDVKENRNLDNVSPKDGSTPGPGEGSQLSNGGGGGPGRKRPLEEGSNGHSKYRLKKRRKTPGPVLPKNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAAEKALRSFVQFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFN GGSGGSGGS MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK* (SEQ ID NO: 83)dsRBD-Linker-hAPOBEC1MDIEDEENMSSSSTDVKENRNLDNVSPKDGSTPGPGEGSQLSNGGGGGPGRKRPLEEGSNGHSKYRLKKRRKTPGPVLPKNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAAEKALRSFVQFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKINPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFN GGSGGSGGS MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR* (SEQ ID NO: 84)MCP-Linker-ADAR1-NESMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGS KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPVGSGSGSLPPLERLTL* (SEQ ID NO: 85) MCP-Linker-ADAR2-NLSMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGS QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTGSGSGSPKKKRKV* (SEQ ID NO: 86) MCP-Linker-ADAR2-NESMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGS QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTGSGSGSLPPLERLTL* (SEQ ID NO: 87) MCP-Linker-rAPOBEC1-NLSMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGSSGSETPGTSESATPES MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFI EKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKGSGSGSPKKKRKV* (SEQ ID NO: 88)MCP-Linker-rAPOBEC1-NESMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGSSGSETPGTSESATPES MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKGSGSGSLPPLERLTL* (SEQ ID NO: 89) MCP-Linker-hAPOBEC1-NLSMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGSSGSETPGTSESATPES MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWRGSGSGSPKKKRKV* (SEQ ID NO: 90) MCP-Linker-hAPOBEC1-NESMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGGSGSGAGSGS PAGGGAPGSGGGSSGSETPGTSESATPES MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWRGSGSGSLPPLERLTL* (SEQ ID NO: 91)Alternate spacer (can be used in place of GGSGGSGGS (SEQ ID NO: 92)):SGSETPGTSESATPES (SEQ ID NO: 93) 3XNLS-4x1N-cdADAR2MPKKKRKVDPKKKRKVDPKKKRKVGSYPYDVPDYAGSNARTRRRERRAEKQAQWKAANGGGGSGGGGSGGGGSNARTRRRERRAEKQAQWKAANGGGGSGGGGSGGGGSNARTRRRERRAEKQAQWKAANGGGGSGGGGSGGGGSNARTRRRE RRAEKQAQWKAANLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP (SEQ ID NO: 94) N22p-hAPOBEC1MPKKKRKVDGSGNARTRRRERRAEKQAQWKAANGGGGTSGSGSGSPAGGGA PGSGGGSMTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR (SEQ ID NO: 95) 3XNLS-4x1N-hAPOBEC1MPKKKRKVDPKKKRKVDPKKKRKVGSYPYDVPDYAGSNARTRRRERRAEKQAQWKAANGGGGSGGGGSGGGGSNARTRRRERRAEKQAQWKAANGGGGSGGGGSGGGGSNARTRRRERRAEKQAQWKAANGGGGSGGGGSGGGGSNARTRRRE RRAEKQAQWKAANMTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR (SEQ ID NO: 96)C-terminal ADAR2 (residues 1-138 deleted)MLRSFVQFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFNLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP*(SEQ ID NO: 97) MS2-RNA:Single:  NNNNNNNNNNNNNNNNNNNNggccAACATGAGGATCACCCATGTCTGCAGggcc(SEQ ID NO: 98) Dual:aACATGAGGATCACCCATGTcNNNNNNNNNNNNNNNNNNNNaACATGAGGATCACCCATGTc (SEQ ID NO: 99) BoxB RNA: Single: NNNNNNNNNNNNNNNNNNNNgggccctgaagaagggccc (SEQ ID NO: 100) Dual:ggGCCCTGAAGAAGGGCccNNNNNNNNNNNNNNNNNNNNNggGCCCTGAAGAAGGGCcc (SEQ ID NO: 101) PP7-RNA:NNNNNNNNNNNNNNNNNNNNccggagcagacgatatggcgtcgctccgg (SEQ ID NO: 102)Dual Hairpin RNA: TGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACNNNNNNNNNNNNNNNNNNNNGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC (SEQ ID NO: 103) A-U to G-C substitutions in adRNAv1: GGGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACCTNNNCNNNNNNNNNNNNNNN (SEQ ID NO: 104)v2: GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACNNNNNNCNNNNNNNNNNNNN (SEQ ID NO: 105) v3:GTGGAAGAGGAGAACAATATGCTAAATGTTGTTCTCGTCTCCCAC NNNNNNCNNNNNNNNNNNNNN(SEQ ID NO: 106) v4:GGGTGGAAGAGGAGAACAATATGCTAAATGTTGTTCTCGTCTCCCACCT NNNCNNNNNNNNNNNNNNN (SEQ ID NO: 107) v5:GGTGAAGAGGAGAACAATATGCTAAATGTTGTTCTCGTCTCCACC NNNNNNCNNNNNNNNNNNNNN (SEQ ID NO: 108) v6:GGTGAAGAGGAGAACAATATGCTAAATGTTGTTCTCGTCTCCACC NNNNNNNCNNNNNNNNNNNNN (SEQ ID NO: 109) v7:GTGGAAGAGGAGAACAATAGGCTAAACGTTGTTCTCGTCTCCCAC NNNNNNCNNNNNNNNNNNNNN (SEQ ID NO: 110) V8:GGGTGGAAGAGGAGAACAATAGGCTAAACGTTGTTCTCGTCTCCCACCT NNNCNNNNNNNNNNNNNNN (SEQ ID NO: 111) v9:GGTGAAGAGGAGAACAATAGCTAAACGTTGTTCTCGTCTCCACC NNNNNNCNNNNNNNNNNNNNN (SEQ ID NO: 112)v10: GGTGAAGAGGAGAACAATAGGCTAAACGTTGTTCTCGTCTCCACCNNNNNNNCNNNNNNNNNNNNN (SEQ ID NO: 113) v11:GGTGTCGAGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCTCGACACC NNNNNNNCNNNNNNNNNN (SEQ ID NO: 114) v12:GGTGTCGAGAACAGCAGAACAATATGCTAAATGTTGTTCTCGTCTCCTCGACACC NNNNNNNCNNNNNNNNNN (SEQ ID NO: 115) v13:GGTGTCGAGAAGAGGAGAACAATAGGCTAAACGTTGTTCTCGTCTCCTCGACACC NNNNNNNCNNNNNNNNNN (SEQ ID NO: 116) dCas9Cj-NES-Linker-cdADAR2(E488Q)MARILAFAIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNYRNKKESYERCIAQSFLKDELKLIFIREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDAIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVHAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKINGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKKSGLPPLERLTLGSGGGG SQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGQGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT (SEQ ID NO: 117) Single and dual ADAR2 recruiting domain:Single: GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACACAAACCGAGCGGTGTCTGT(SEQ ID NO: 118) Dual 1:GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACCAAACCGAGCGGTGTCTGTGGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC (SEQ ID NO: 119) Dual 2:GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACTACAAACCGAGCGGTGTCTGGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC (SEQ ID NO: 120) Dual 3:GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACTTTACAAACCGAGCGGTGTCGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC (SEQ ID NO: 121) Dual 4:GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACGTTTTACAAACCGAGCGGTGGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC(SEQ ID NO: 122) Dual 5:GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACAAGTTTTACAAACCGAGCGGGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC (SEQ ID NO: 123)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the vector constructs developed for thedelivery of the modified endogenous or orthogonal tRNA.

FIG. 2A-B show suppression efficiencies of the tRNA constructs: (FIG.2A) Relative efficiencies of the suppressor tRNAs derived from arginine,serine and leucine towards the amber, ochre and opal stop codons;Representative images showing the restoration of GFP expression in thepresence of the Ser tRNAAmber (FIG. 2B) Comparison of the suppressionefficiencies of the single or dual pyrrolysyl tRNAs towards amber, ochreand opal stop codons in the presence of 2 mM UAA; Representative imagesshowing the relative GFP restoration using single and dual pyrrolysyltRNAAmber in the presence of 2 mM UAA.

FIG. 3 shows the GFP reporter results for dystrophin with various tRNAand amino acids.

FIG. 4 shows the results of the dystrophin restoration experimentsperformed in mdx mice.

FIG. 5 shows sequences used to generate the ADAR2 constructs (SEQ ID NOS164-166, respectively, in order of appearance).

FIG. 6 shows non-limiting examples of RNA level point mutations to acodon that can be made by ADAR2.

FIG. 7 shows exemplary schematics of constructs that may be used in anADAR2 based RNA editing system.

FIG. 8 shows the results of optimization of the length of adRNA anddistance of the edit from the ADAR2 recruiting domain. The first numberin the shorthand for each category on the Y-axis is the length of adRNAand the second number (following the dash) is the distance of edit fromADAR2 recruiting domain. 20-6 with ADAR2 recruiting region v2 gave usthe best results.

FIG. 9 shows in vitro restoration of GFP expression using the editingsystems described herein.

FIG. 10 shows the results of optimization of hairpins with mismatches(SEQ ID NOS 167-172, respectively, in order of appearance). The firstnumber in the shorthand for each category on the Y-axis is the number ofmismatches and the second number is the number of bases it is from thetarget. For example, 13 is 1 mismatch, 3 bases away from the target.

FIG. 11 shows the results of varying lengths of toe hold, guide RNAsequences with no mismatches to the target.

FIG. 12A-C show results of (FIG. 12A) immunostaining, (FIG. 12B) Westernblot, and (FIG. 12C) in vitro OTC mRNA editing assays (SEQ ID NOS173-174, respectively, in order of appearance).

FIG. 13 is a Western blot that shows the restoration of dystrophinexpression using suppressor tRNA, in comparison with the Cas9 basedapproaches.

FIG. 14 shows normalized dystrophin mRNA levels.

FIG. 15 shows results of immunostaining.

FIG. 16A-D shows in vitro suppression and editing of stop codons in GFPreporter mRNA: (FIG. 16A) Activity of arginine, serine and leucinesuppressor tRNAs targeting amber, ochre and opal stop codons (n=3independent replicates). (FIG. 16B) Orthogonal tRNA/aaRS (MbPylRS) basedsuppression of amber, ochre and opal stop codons in the presence of oneor two copies of the pyrrolysyl-tRNA delivered via an AAV vector and inthe presence of 1 mM NE-Boc-L-Lysine (n=3 independent replicates)(p-values 0.022, 0.002, 0.027 respectively). (FIG. 16C) ADAR2 based RNAediting efficiencies of amber and ochre stop codons, in one-step,two-steps, or in combination with suppressor tRNAs (n=3 independentreplicates). (FIG. 16D) ADAR2 based RNA editing efficiencies of amberand ochre stop codons in the presence of one or two copies of the adRNA,delivered via an AAV vector (n=3 or 6 independent replicates) (p-values0.0003, 0.0001, 0.0015 respectively).

FIG. 17A-E shows in vivo RNA targeting in mouse models of human disease:(FIG. 17A) Schematic of the DNA and RNA targeting approaches to restoredystrophin expression in mdx mice: (i) a dual gRNA-CRISPR based approachleading to in frame excision of exon 23; (ii) tRNA suppression of theochre codon; and (iii) ADAR2 based editing of the ochre codon. (FIG.17B) Immunofluorescence staining for dystrophin and nNOS in controls andtreated samples (scale bar: 250 μm). (FIG. 17C) In vivo TAA→TGG/TAG/TGARNA editing efficiencies in corresponding treated adult mdx mice (n=3 or4 mice). (FIG. 17D) Schematic of the OTC locus in spf^(ash) mice whichhave a G→A point mutation at a donor splice site or missense in the lastnucleotide of exon 4, and approach for correction of mutant OTC mRNA viaADAR2 mediated RNA editing (FIG. 17E) In vivo A→G RNA editingefficiencies in corresponding treated adult spf^(ash) mice (n=3 or 4mice).

FIG. 18A-B show in vitro tRNA suppression evaluation and optimization:(FIG. 18A) Specificity of modified serine suppressor tRNAs for ochre andopal stop codons (n=3 independent replicates). (FIG. 18B) Ochre stopcodon suppression efficiency utilizing three different aaRS: MbPylRS,MmPylRS and AcKRS, and two or four copies of the pyrroysyl-tRNA, orserine suppressor tRNA, all delivered using an AAV vector. MbPylRS,MmPylRS: 1 mM NE-Boc-L-Lysine; AcKRS: 1 or 10 mM Nε-Acetyl-L-Lysine (n=3independent replicates).

FIG. 19A-C shows in vitro ADAR2 mediated site-specific RNA editingevaluation and optimization: (FIG. 19A) GFP expression is restored whenadRNA/radRNA has two mismatches corresponding to the two adenosines inthe ochre stop codon. Presence of a single mismatch results in theformation of an amber or opal stop codon (n=3 independent replicates)(SEQ ID NOS 175-179, respectively, in order of appearance). (FIG. 19B)Panel of adRNA designs used (SEQ ID NOS 180-181, respectively, in orderof appearance). (FIG. 19C) Optimization of adRNA antisense region usingadRNA design 1: length and distance from the ADAR2 recruiting regionwere systematically varied, and editing efficiency calculated as a ratioof Sanger peak heights G/(A+G) (n=3 independent replicates) (SEQ ID NOS182-206, respectively, in order of appearance).

FIG. 20A-C shows in vivo targeting of dystrophin mRNA via suppressortRNAs: (FIG. 20A) Progressively increasing restoration of dystrophinexpression over time in mdx mice treated withAAV8-dual-serine-ochre-tRNA. (FIG. 20B) UAA inducible nNOS localizationin mdx mice treated with AAV8-dual-pyrrolysine-ochre-tRNA-MbPylRS. (FIG.20C) Western blot for dystrophin shows partial recovery of dystrophinexpression in the mdx mice treated with a serine tRNA ochre, thepyrrolysyl-tRNA ochre and administered with the UAA, as well as inCas9/gRNAs treated samples.

FIG. 21A-D show in vitro and in vivo editing of dystrophin and OTC mRNA:(FIG. 21A) Representative Sanger sequencing plot showing 12.7% editingof the ochre stop codon (TAA→TGG) in a fragment of the mdx dystrophinmRNA expressed in HEK 293T cells (quantified using NGS) (SEQ ID NOS207-208, respectively, in order of appearance). (FIG. 21B)Representative example of in vivo RNA editing analyses of treated mdxmouse (quantified using NGS) (SEQ ID NOS 209-216, respectively, in orderof appearance). (FIG. 21C) Representative Sanger sequencing plot showing29.7% correction of the point mutation in a fragment of the spf^(ash)OTC mRNA expressed in HEK 293T cells (quantified using NGS) (SEQ ID NOS217-218, respectively, in order of appearance). (FIG. 21D)Representative example of in vivo RNA editing analyses of treatedspf^(ash) mouse (quantified using NGS) (SEQ ID NOS 219-226,respectively, in order of appearance).

FIG. 22A-B show in vitro editing efficiency of ADAR2-E488Q. ADAR2-E488Qenables higher efficiency than the ADAR2 in the in vitro editing of:(FIG. 22A) a fragment of spf^(ash) OTC mRNA expressed in HEK293T cells(n=3 independent replicates) (p-value 0.037), and (FIG. 22B) a fragmentof mdx dystrophin mRNA expressed in HEK293T cells (n=3 independentreplicates) (p-values 0.048, 0.012 respectively). Efficiency wascalculated as a ratio of Sanger peak heights G/(A+G).

FIG. 23A-D show schematics of (FIG. 23A) MCP or N22 fusions with ADAR1or ADAR2, (FIG. 23B) recruitment of APOBEC by adRNA, (FIG. 23C) a moregeneral adRNA architecture, and (FIG. 23D) the structure of the v2 adRNAscaffold after folding (SEQ ID NO: 227).

FIG. 24A-B show schematics of optional embodiments in which (FIG. 24A)endogenous ADAR2 can be used in the methods disclosed herein in tissueswith high endogenous ADAR2, e.g., brain, lung, and spleen and (FIG. 24B)ADAR1 and/or ADAR2 levels can be increased in tissues with low levels ofendogenous ADAR1 and ADAR2. Clockwise from the left, (1) delivery ofadRNA and ADAR2 would result in high levels of RNA editing, (2) deliveryof adRNA alone is likely to bring about little or no editing due to thelow levels of endogenous ADAR1 and ADAR2, (3) treatment of cells withIFNs will lead to an increase in the ADAR1 (p150) levels but is unlikelyto bring about any editing of the RNA target in the absence of theadRNA; (4) treatment of cells with IFNs with the addition of adRNA willlead to elevated levels of ADAR1 (p150) and in the presence of adRNA, islikely to lead to high levels of target RNA editing, (5) treatment ofcells with IFNs with the addition of adRNA and ADAR2 will lead toelevated levels of ADAR1 expression, and high levels of RNA editing.

FIG. 25 shows the rate of UAA to UAG conversion. The UAA is converted toUAG via ADAR2 based editing and addition of suppressor tRNA targetingthe UAG stop codon led to partial restoration of GFP expression

FIG. 26 shows the results of in vivo RNA editing in the mdx mouse modelof muscular dystrophy.

FIG. 27 shows the resulting edited sequences resulting from use of thepromiscuous C-terminal ADAR2 (SEQ ID NOS 228-264, respectively, in orderof appearance).

FIG. 28 shows editing efficiency of the stabilized scaffolds (SEQ ID NOS104-113, respectively, in order of appearance).

FIG. 29 shows the fraction of edited mRNA with single versus dual ADAR2recruiting domains and the corresponding sequences (SEQ ID NOS 118-123,respectively, in order of appearance).

FIG. 30 shows the fraction of edited mRNA with various MCP-ADARscaffolds (SEQ ID NOS 265-269, respectively, in order of appearance).

FIG. 31 shows alternative splice variants of OTC and is taken fromHodges, P. E. & Rosenberg, L. E. The spfash mouse: a missense mutationin the ornithine transcarbamylase gene also causes aberrant mRNAsplicing. Proc. Natl. Acad. Sci. U.S.A. 86, 4142-4146 (1989) (SEQ ID NOS270-275, respectively, in order of appearance).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All nucleotide sequencesprovided herein are presented in the 5′ to 3′ direction. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods, devices, and materials are now described. Alltechnical and patent publications cited herein are incorporated hereinby reference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The practice of the present technology will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology, and recombinant DNA,which are within the skill of the art. See, e.g., Sambrook and Russelleds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); and Herzenberg et al. eds (1996) Weir's Handbook ofExperimental Immunology.

The terminology used in the description herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. All publications, patent applications,patents and other references mentioned herein are incorporated byreference in their entirety.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate oralternatively by a variation of +/−15%, or alternatively 10% oralternatively 5% or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the disclosure also contemplates that in someembodiments, any feature or combination of features set forth herein canbe excluded or omitted. To illustrate, if the specification states thata complex comprises components A, B and C, it is specifically intendedthat any of A, B or C, or a combination thereof, can be omitted anddisclaimed singularly or in any combination.

Unless explicitly indicated otherwise, all specified embodiments,features, and terms intend to include both the recited embodiment,feature, or term and biological equivalents thereof.

Definitions

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a polypeptide” includes a plurality ofpolypeptides, including mixtures thereof.

The term “about,” as used herein when referring to a measurable valuesuch as an amount or concentration and the like, is meant to encompassvariations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specifiedamount.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

A “subject” of diagnosis or treatment is a cell or an animal such as amammal, or a human. Non-human animals subject to diagnosis or treatmentand are those subject to infections or animal models, for example,simians, murines, such as, rats, mice, chinchilla, canine, such as dogs,leporids, such as rabbits, livestock, sport animals, and pets.

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense to refer to a compound of two or moresubunit amino acids, amino acid analogs or peptidomimetics. The subunitsmay be linked by peptide bonds. In another embodiment, the subunit maybe linked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics. As used herein, the term “fusion protein” refers to aprotein comprised of domains from more than one naturally occurring orrecombinantly produced protein, where generally each domain serves adifferent function. In this regard, the term “linker” refers to aprotein fragment that is used to link these domains together—optionallyto preserve the conformation of the fused protein domains and/or preventunfavorable interactions between the fused protein domains which maycompromise their respective functions.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of this invention that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. In some embodiments, thepolynucleotide may comprise one or more other nucleotide bases, such asinosine (I), a nucleoside formed when hypoxanthine is attached toribofuranose via a β-N9-glycosidic bond, resulting in the chemicalstructure:

Inosine is read by the translation machinery as guanine (G). The term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.

The terms “equivalent” or “biological equivalent” are usedinterchangeably when referring to a particular molecule, biological, orcellular material and intend those having minimal homology while stillmaintaining desired structure or functionality.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

As used herein, the term “functional” may be used to modify anymolecule, biological, or cellular material to intend that itaccomplishes a particular, specified effect.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease, disorder, or condition or sign orsymptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure for a disorder and/or adverse effect attributable to thedisorder.

“Administration” can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents are known in the art. Route ofadministration can also be determined and method of determining the mosteffective route of administration are known to those of skill in the artand will vary with the composition used for treatment, the purpose ofthe treatment, the health condition or disease stage of the subjectbeing treated, and target cell or tissue. Non-limiting examples of routeof administration include oral administration, nasal administration,injection, and topical application.

The term “effective amount” refers to a quantity sufficient to achieve adesired effect. In the context of therapeutic or prophylacticapplications, the effective amount will depend on the type and severityof the condition at issue and the characteristics of the individualsubject, such as general health, age, sex, body weight, and tolerance topharmaceutical compositions. In the context of an immunogeniccomposition, in some embodiments the effective amount is the amountsufficient to result in a protective response against a pathogen. Inother embodiments, the effective amount of an immunogenic composition isthe amount sufficient to result in antibody generation against theantigen. In some embodiments, the effective amount is the amountrequired to confer passive immunity on a subject in need thereof. Withrespect to immunogenic compositions, in some embodiments the effectiveamount will depend on the intended use, the degree of immunogenicity ofa particular antigenic compound, and the health/responsiveness of thesubject's immune system, in addition to the factors described above. Theskilled artisan will be able to determine appropriate amounts dependingon these and other factors.

In the case of an in vitro application, in some embodiments theeffective amount will depend on the size and nature of the applicationin question. It will also depend on the nature and sensitivity of the invitro target and the methods in use. The skilled artisan will be able todetermine the effective amount based on these and other considerations.The effective amount may comprise one or more administrations of acomposition depending on the embodiment.

The term “Cas9” refers to a CRISPR associated endonuclease referred toby this name (for example, UniProtKB G3ECR1 (CAS9_STRTR)) as well asdead Cas9 or dCas9, which lacks endonuclease activity (e.g., withmutations in both the RuvC and HNH domain). The term “Cas9” may furtherrefer to equivalents of the referenced Cas9 having at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto, includingbut not limited to other large Cas9 proteins. In some embodiments, theCas9 is derived from Campylobacter jejuni or another Cas9 orthologue1000 amino acids or less in length.

The term “vector” refers to a polynucleotide (usually DNA) used toartificially carry foreign genetic material to another cell where it canbe replicated or expressed. Non-limiting exemplary vectors includeplasmids, viral vectors, cosmids, and artificial chromosomes. Suchvectors may be derived from a variety of sources, including bacterialand viral sources. A non-limiting exemplary viral source for a plasmidis adeno-associated virus.

As used herein, the term “recombinant expression system” refers to agenetic construct or constructs for the expression of certain geneticmaterial formed by recombination; the term “construct” in this regard isinterchangeable with the term “vector” as defined herein.

The term “adeno-associated virus” or “AAV” as used herein refers to amember of the class of viruses associated with this name and belongingto the genus dependoparvovirus, family Parvoviridae. Multiple serotypesof this virus are known to be suitable for gene delivery; all knownserotypes can infect cells from various tissue types. At least 11,sequentially numbered, are disclosed in the prior art. Non-limitingexemplary serotypes useful for the purposes disclosed herein include anyof the 11 serotypes, e.g., AAV2 and AAV8.

The term “lentivirus” as used herein refers to a member of the class ofviruses associated with this name and belonging to the genus lentivirus,family Retroviridae. While some lentiviruses are known to causediseases, other lentivirus are known to be suitable for gene delivery.See, e.g., Tomás et al. (2013) Biochemistry, Genetics and MolecularBiology: “Gene Therapy—Tools and Potential Applications,” ISBN978-953-51-1014-9, DOI: 10.5772/52534.

As used herein the term “restoring” in relation to expression of aprotein refers to the ability to establish expression of full lengthprotein where previously protein expression was truncated due tomutation.

The term “mutation” as used herein, refers to an alteration to a nucleicacid sequence encoding a protein relative to the consensus sequence ofsaid protein. “Missense” mutations result in the substitution of onecodon for another; “nonsense” mutations change a codon from one encodinga particular amino acid to a stop codon. Nonsense mutations often resultin truncated translation of proteins. “Silent” mutations are those whichhave no effect on the resulting protein. As used herein the term “pointmutation” refers to a mutation affecting only one nucleotide in a genesequence. “Splice site mutations” are those mutations present pre-mRNA(prior to processing to remove introns) resulting in mistranslation andoften truncation of proteins from incorrect delineation of the splicesite.

“Messenger RNA” or “mRNA” is a nucleic acid molecule that is transcribedfrom DNA and then processed to remove non-coding sections known asintrons. The resulting mRNA is exported from the nucleus (or anotherlocus where the DNA is present) and translated into a protein. The term“pre-mRNA” refers to the strand prior to processing to remove non-codingsections.

“Transfer ribonucleic acid” or “tRNA” is a nucleic acid molecule thathelps translate mRNA to protein. tRNA have a distinctive foldedstructure, comprising three hairpin loops; one of these loops comprisesa “stem” portion that encodes an anticodon. The anticodon recognizes thecorresponding codon on the mRNA. Each tRNA is “charged with” an aminoacid corresponding to the mRNA codon; this “charging” is accomplished bythe enzyme tRNA synthetase. Upon tRNA recognition of the codoncorresponding to its anticodon, the tRNA transfers the amino acid withwhich it is charged to the growing amino acid chain to form apolypeptide or protein. Endogenous tRNA can be charged by endogenoustRNA synthetase. Accordingly, endogenous tRNA are typically charged withcanonical amino acids. Orthogonal tRNA, derived from an external source,require a corresponding orthogonal tRNA synthetase. Such orthogonaltRNAs may be charged with both canonical and non-canonical amino acids.In some embodiments, the amino acid with which the tRNA is charged maybe detectably labeled to enable detection in vivo. Techniques forlabeling are known in the art and include, but are not limited to, clickchemistry wherein an azide/alkyne containing unnatural amino acid isadded by the orthogonal tRNA/synthetase pair and, thus, can be detectedusing alkyne/azide comprising fluorophore or other such molecule.

The term “stop codon” intends a three nucleotide contiguous sequencewithin messenger RNA that signals a termination of translation.Non-limiting examples include in RNA, UAG, UAA, UGA and in DNA TAG, TAAor TGA. Unless otherwise noted, the term also includes nonsensemutations within DNA or RNA that introduce a premature stop codon,causing any resulting protein to be abnormally shortened. tRNA thatcorrespond to the various stop codons are known by specific names: amber(UAG), ochre (UAA), and opal (UGA).

“Canonical amino acids” refer to those 20 amino acids found naturally inthe human body shown in the table below with each of their three letterabbreviations, one letter abbreviations, structures, and correspondingcodons:

non-polar, aliphatic residues Glycine Gly G

GGU GGC GGA GGG Alanine Ala A

GCU GCC GCA GCG Valine Val V

GUU GUC GUA GUG Leucine Leu L

UUA UUG CUU CUC CUA CUG Isoleucine Ile I

AUU AUC AUA Proline Pro P

CCU CCC CCA CCG aromatic residues Phenylalanine Phe F

UUU UUC Tyrosine Tyr W

UAU UAC Tryptophan Trp W

UGG polar, non-charged residues Serine Ser S

UCU UCC UCA UCG AGU AGC Threonine Thr T

ACU ACC ACA ACG Cysteine Cys C

UGU UGC Methionine Met M

AUG Asparagine Asn N

AAU AAC Glutamine Gln Q

CAA CAG positively charged residues Lysine Lys K

AAA AAG Arginine Arg R

CGU CGC CGA CGG AGA AGG Histidine His H

CAU CAC negatively charged residues Aspartate Asp D

GAU GAC Glutamate Glu E

GAA GAG

The term “non-canonical amino acids” refers to those synthetic orotherwise modified amino acids that fall outside this group, typicallygenerated by chemical synthesis or modification of canonical amino acids(e.g. amino acid analogs). The present disclosure employs proteinogenicnon-canonical amino acids in some of the methods and vectors disclosedherein. A non-limiting exemplary non-canonical amino acid is pyrrolysine(Pyl or O), the chemical structure of which is provided below:

Inosine (I) is another exemplary non-canonical amino acid, which iscommonly found in tRNA and is essential for proper translation accordingto “wobble base pairing.” The structure of inosine is provided above.

The term “ADAR” as used herein refers to an adenosine deaminase that canconvert adenosines (A) to inosines (I) in an RNA sequence. ADAR1 andADAR2 are two exemplary species of ADAR that are involved in mRNAediting in vivo. Non-limiting exemplary sequences for ADAR1 may be foundunder the following reference numbers: HGNC: 225; Entrez Gene: 103;Ensembl: ENSG 00000160710; OMIM: 146920; UniProtKB: P55265; andGeneCards: GC01M154554, as well as biological equivalents thereof.Non-limiting exemplary sequences for ADAR2 may be found under thefollowing reference numbers: HGNC: 226; Entrez Gene: 104; Ensembl:ENSG00000197381; OMIM: 601218; UniProtKB: P78563; and GeneCards:GC21P045073, as well as biological equivalents thereof. Furthernon-limited exemplary sequences of the catalytic domain are providedhereinabove. The forward and reverse RNA used to direct site-specificADAR editing are known as “adRNA” and “radRNA,” respectively. Thecatalytic domains of ADAR1 and ADAR2 are comprised in the sequencesprovided herein below.

ADAR1 catalytic domain:

(SEQ ID NO: 124) KAERMGFTEVTPVTGASLRRTMLLLSRSPEAQPKTLPLTGSTFHDQIAMLSHRCFNTLTNSFQPSLLGRKILAAIIMKKDSEDMGVVVSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNFYLCPV

ADAR2 catalytic domain:

(SEQ ID NO: 125) QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT

The double stranded RNA binding domains (dsRBD) of an ADAR is comprisedin the sequence provided herein below.

ADAR dsRBD:

(SEQ ID NO: 126) MDIEDEENMSSSSTDVKENRNLDNVSPKDGSTPGPGEGSQLSNGGGGGPGRKRPLEEGSNGHSKYRLKKRRKTPGPVLPKNALMQLNEIKPGLQYTLLSQTGPVHAPLFVMSVEVNGQVFEGSGPTKKKAKLHAAEKALRSFVQFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFN

It is appreciated that further mutations can be made to the sequence ofthe ADAR and/or its various domains. For example, Applicants havegenerated E488Q and E1008Q mutants of both ADAR1 and ADAR2, as well as a“promiscuous” variant of ADAR2—resulting from a C-terminal deletion.This “promiscuous” variant is known as such because it demonstratedpromiscuity in edited reads with several As close to a target sequenceshowing an A to G conversion (verified across 2 different loci). Thesequence of this variant is provided herein below.

“Promiscuous” ADAR2 variant:

(SEQ ID NO: 127) MLRSFVQFPNASEAHLAMGRTLSVNTDFTSDQADFPDTLFNGFETPDKAEPPFYVGSNGDDSFSSSGDLSLSASPVPASLAQPPLPVLPPFPPPSGKNPVMILNELRPGLKYDFLSESGESHAKSFVMSVVVDGQFFEGSGRNKKLAKARAAQSALAAIFNLHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVVMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFS LTP*Not to be bound by theory, a C-terminal deletion in ADAR1 may producethe same or similar effect.

The term “deficiency” as used herein refers to lower than normal(physiologically acceptable) levels of a particular agent. In context ofa protein, a deficiency refers to lower than normal levels of the fulllength protein.

The term “dystrophin” as used herein refers to the protein correspondingwith that name and encoded by the gene Dmd; a non-limiting example ofwhich is found under UniProt Reference Number P11532 (for humans) andP11531 (for mice).

The term “ornithine transcarbamylase” or “OTC” as used herein refers tothe protein corresponding with that name and encoded by the gene Otc; anon-limiting example of which is found under UniProt Reference NumberP00480 (for humans) and P11725 (for mice). OTC deficiency is an X-linkedgenetic condition resulting in high concentrations of ammonia in blood.In some cases, OTC deficiency is caused by a G→A splice site mutation inthe donor splice site of exon 4 that results in mis-splicing of thepre-mRNA. This mutation results in the formation of a protein thateither is elongated or bears a point mutation. There is a 15-20 foldreduction in the OTC protein levels. The FIG. 31 (taken from Hodges, P.E. & Rosenberg, L. E. The spfash mouse: a missense mutation in theornithine transcarbamylase gene also causes aberrant mRNA splicing.Proc. Natl. Acad. Sci. U.S.A. 86, 4142-4146 (1989)) shows thealternative forms produced. The sequences thereof are provided below:

OTC pre-mRNA (wild type): . . .CTCACAGACACCGCTC GGTTTGTAAAACTTTTCTTC. . . (SEQ ID NO: 128)OTC pre-mRNA(mutant): . . .CTCACAGACACCGCTC

GTTTGTAAAACTTTTCTTC. . . (SEQ ID NO: 129)OTC mRNA (incorrectly spliced, mutant): . . .CTCACAGACACCGCTC AGTTTGTAAAACTTTTCTTC. . . (SEQ ID NO: 130)OTC mRNA (correctly spliced, mutant): . . .CTCACAGACACCGCTC ATGTCTTATCTAGCATGACA. . . (SEQ ID NO: 131)OTC mRNA (correctly spliced, wild type): . . .CTCACAGACACCOCTC GTGTCTTATCTAGCATGACA. . . (SEQ ID NO: 132)As shown above, a correct splice variant may be produced when themutation is present; however, such production results in a missensemutation, which also can contribute to OTC deficiency.

The terms “hairpin,” “hairpin loop,” “stem loop,” and/or “loop” usedalone or in combination with “motif” is used in context of anoligonucleotide to refer to a structure formed in single strandedoligonucleotide when sequences within the single strand which arecomplementary when read in opposite directions base pair to form aregion whose conformation resembles a hairpin or loop.

As used herein, the term “domain” refers to a particular region of aprotein or polypeptide and is associated with a particular function. Forexample, “a domain which associates with an RNA hairpin motif” refers tothe domain of a protein that binds one or more RNA hairpin. This bindingmay optionally be specific to a particular hairpin. For example, the M2bacteriophage coat protein (MCP) is capable of specifically binding toparticular stem-loop structures, including but not limited to the MS2stem loop. See, e.g. Peabody, D. S., “The RNA binding site ofbacteriophage MS2 coat protein.” EMBO J. 12(2):595-600 (1993); Corriganand Chubb, “Biophysical Methods in Cell Biology” Methods in Cell Biology(2015). Similarly, λ N22—referred to herein as “N22 peptide” is capableof specifically binding to particular stem-loop structures, includingbut not limited to BoxB stem loops. See, e.g., Cilley and Williamson,“Analysis of bacteriophage N protein and peptide binding to boxB RNAusing polyacrylamide gel coelectrophoresis (PACE).” RNA 3(1):57-67(1997). The sequences of both MCP and MS2 stem loop and N22 peptide andBoxB loop are provided hereinabove in context of fusion proteins with anADAR (MCP, N22 peptide) and use in adRNA (MS2 stem loop, BoxB loop),respectively.

The term “APOBEC” as used herein refers to any protein that falls withinthe family of evolutionarily conserved cytidine deaminases involved inmRNA editing—catalyzing a C to U conversion—and equivalents thereof. Insome aspects, the term APOBEC refers to any one of APOBEC1, APOBEC2,APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H,APOBEC4, or equivalents each thereof. Non-limiting exemplary sequencesof fusion proteins comprising one or more APOBEC domains are providedherein both fused to an ADAR domain or fused to alternative domains torender them suitable for use in an RNA editing system. To this end,APOBECs can be considered an equivalent of ADAR—catalyzing editingalbeit by a different conversion. Thus, not to be bound by theory,Applicants believe that all embodiments contemplated herein for use withan ADAR based editing system may be adapted for use in an APOBEC basedRNA editing system.

As used herein, the term “interferon” refers to a group of signalingproteins known to be associated with the immune response. In context ofthis application, the interferons of interest are those that result inenhanced expression of an ADAR. The correlation between interferon α andADAR1 is well known, and, thus, the present disclosure contemplates useof interferon α as a means of increasing endogenous ADAR1 expression.Commercial sources of isolated or recombinant interferon α include butare not limited to Sigma-Aldrich, R&D Systems, Abcam, and Thermo FisherScientific. Alternatively, interferon α may be produced using a knownvector and given protein sequence, e.g. Q6QNB6 (human IFNA).

It is to be inferred without explicit recitation and unless otherwiseintended, that when the present disclosure relates to a polypeptide,protein, polynucleotide or antibody, an equivalent or a biologicallyequivalent of such is intended within the scope of this disclosure. Asused herein, the term “biological equivalent thereof” is intended to besynonymous with “equivalent thereof” when referring to a referenceprotein, antibody, polypeptide or nucleic acid, intends those havingminimal homology while still maintaining desired structure orfunctionality. Unless specifically recited herein, it is contemplatedthat any polynucleotide, polypeptide or protein mentioned herein alsoincludes equivalents thereof. For example, an equivalent intends atleast about 70% homology or identity, or at least 80% homology oridentity and alternatively, or at least about 85%, or alternatively atleast about 90%, or alternatively at least about 95%, or alternatively98% percent homology or identity and exhibits substantially equivalentbiological activity to the reference protein, polypeptide or nucleicacid. Alternatively, when referring to polynucleotides, an equivalentthereof is a polynucleotide that hybridizes under stringent conditionsto the reference polynucleotide or its complement.

Applicants have provided herein the polypeptide and/or polynucleotidesequences for use in gene and protein transfer and expression techniquesdescribed below. It should be understood, although not always explicitlystated that the sequences provided herein can be used to provide theexpression product as well as substantially identical sequences thatproduce a protein that has the same biological properties. These“biologically equivalent” or “biologically active” polypeptides areencoded by equivalent polynucleotides as described herein. They maypossess at least 60%, or alternatively, at least 65%, or alternatively,at least 70%, or alternatively, at least 75%, or alternatively, at least80%, or alternatively at least 85%, or alternatively at least 90%, oralternatively at least 95% or alternatively at least 98%, identicalprimary amino acid sequence to the reference polypeptide when comparedusing sequence identity methods run under default conditions. Specificpolypeptide sequences are provided as examples of particularembodiments. Modifications to the sequences to amino acids withalternate amino acids that have similar charge. Additionally, anequivalent polynucleotide is one that hybridizes under stringentconditions to the reference polynucleotide or its complement or inreference to a polypeptide, a polypeptide encoded by a polynucleotidethat hybridizes to the reference encoding polynucleotide under stringentconditions or its complementary strand. Alternatively, an equivalentpolypeptide or protein is one that is expressed from an equivalentpolynucleotide.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PC reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubationtemperatures of about 25° C. to about 37° C.; hybridization bufferconcentrations of about 6×SSC to about 10×SSC; formamide concentrationsof about 0% to about 25%; and wash solutions from about 4×SSC to about8×SSC. Examples of moderate hybridization conditions include: incubationtemperatures of about 40° C. to about 50° C.; buffer concentrations ofabout 9×SSC to about 2×SSC; formamide concentrations of about 30% toabout 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples ofhigh stringency conditions include: incubation temperatures of about 55°C. to about 68° C.; buffer concentrations of about 1×SSC to about0.1×SSC; formamide concentrations of about 55% to about 75%; and washsolutions of about 1×SSC, 0.1×SSC, or deionized water. In general,hybridization incubation times are from 5 minutes to 24 hours, with 1,2, or more washing steps, and wash incubation times are about 1, 2, or15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It isunderstood that equivalents of SSC using other buffer systems can beemployed.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, or alternatively less than 25% identity, withone of the sequences of the present invention.

Modes of Carrying Out the Disclosure

Point mutations underlie many genetic diseases. In this regard, whileprogrammable DNA nucleases have been used to repair mutations, their usefor gene therapy poses multiple challenges: one, efficiency ofhomologous recombination is typically low in cells; two, an activenuclease presents a risk of introducing permanent off-target mutations;and three, prevalent programmable nucleases typically comprise elementsof non-human origin raising the potential of in vivo immunogenicity. Inlight of these, approaches to instead directly target RNA, and use ofmolecular machinery native to the host would be highly desirable.Towards this, Applicants have engineered and optimized two complementaryapproaches, referred together hereon as tRiAD, based on the use of tRNAsin codon suppression and adenosine deaminases in RNA editing.Specifically, by delivering modified endogenous tRNAs or the RNA editingenzyme ADAR and an associated guiding RNA (adRNA) via adeno-associatedviruses, Applicants enabled premature stop codon read-through andcorrection in the mdx mouse model of muscular dystrophy that harbors anonsense mutation in the dystrophin gene. Additionally, Applicantsengineered ADAR2 mediated correction of a point mutation in liver RNA ofthe spf^(ash) mouse model of ornithine transcarbamylase (OTC)deficiency. Taken together, the results disclosed herein establish theuse of suppressor tRNAs and ADAR2 for in vivo RNA targeting, and thisintegrated tRiAD approach is robust, genomically scarless, andpotentially non-immunogenic, as it utilizes effector RNAs and humanproteins.

Aspects of the disclosure relate to a tRNA based protein editing systemoptionally alone or in combination with an ADAR based RNA editing systemcomprising one or more forward guide RNAs for the ADAR (“adRNAs”) andone or more corresponding reverse guide RNAs for the ADAR (“radRNAs”) tothe subject, wherein the ADAR based RNA editing system specificallyedits a point mutation in an RNA sequence encoding a gene.

The tRNA based protein editing system may comprise endogenous modifiedtRNA and/or orthogonal tRNA in order to prevent off target editing ofproteins. In this regard, systems for the control of these tRNA aredisclosed herein below.

The adRNA architecture for use in the ADAR based RNA editing system isrelatively simple, comprising a RNA targeting domain, complementary tothe target and, optionally, one or two recruiting domains (also referredto as aptamers) that recruit RNA binding domains of various proteins.The optional recruiting domains are positioned at the 5′ and/or 3′ endsof the RNA targeting domain. A schematic of adRNA bound to its mRNAtarget is provided in FIG. 23C. In some embodiments, the adRNA featuresan A-C mismatch, which prompts editing function of the ADAR. A similarframework can be used to target pre-mRNA, prior to intron processing byadapting the scaffold to target the pre-mRNA present in the nucleus.This approach is taken in the non-limiting exemplary methods involvingOTC deficiency—involving a splice site mutation, whereas an mRNA editingapproach is taken in the non-limiting exemplary methods involvingdystrophin deficiency—involving a nonsense mutation.

Applicants tested a series of scaffolds, shown in FIG. 19C, to recruitRNA binding domains of the ADARs. The sequences provided in the figurerepresent the recruiting domain and the italicized Ns represent thenucleotides complimentary to the target. The C is the mismatch thatprompts the editing function. Sequences of varying length and mismatchposition may be tested to determine the best adRNA for the desiredtarget. For example, residues in the recruiting domain of the adRNAsgenerated by Applicants were modified as follows (5′-3′):

v1: (SEQ ID NO: 104) GGGTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACCTNNN C NNNNNNNNNNNNNNN  v2: (SEQ ID NO: 105)GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC NNNNNN C NNNNNNNNNNNNN v3: (SEQ ID NO: 106) GTGGAAGAGGAGAACAATATGCTAAATGTTGTTCTCGTCTCCCAC NNNNNN C NNNNNNNNNNNNNN  v4: (SEQ ID NO: 107)GGGTGGAAGAGGAGAACAATATGCTAAATGTTGTTGTTCTCGTCTCCC  ACCT NNN CNNNNNNNNNNNNNNN  v5: (SEQ ID NO: 108)GGTGAAGAGGAGAACAATATGCTAAATGTTCTCGTCTCCACC NNNNN  N C NNNNNNNNNNNNNN v6: (SEQ ID NO: 109) GGTGAACAGCACAACAATATGCTAAATGTTGTTCTCGTCTCCACC NNNNNNN C NNNNNNNNNNNNN  v7: (SEQ ID NO: 110)GTGGAAGAGGAGAACAATAGGCTAAACGTTGTTCTCGTCTCCCAC  NNNNNN C NNNNNNNNNNNNNNv8: GGGTGGAAGAGGAGAACAATAGGCTAAACGTTGTTCTCGTCTCCCACCT   NNN CNNNNNNNNNNNNNNN(SEQ ID NO: 111) v9: (SEQ ID NO: 112)GGTGAACAGCACAACAATAGGCTAAACGTTGTTCTCGTCTCCACC  NNNNNN C NNNNNNNNNNNNNN v10: (SEQ ID NO: 113) GGTGAAGAGCAGAACAATAGGCTAAACGTTGTTCTCGTCTCCACC NNNNNNN C NNNNNNNNNNNNN v11: (SEQ ID NO: 114)GGTGTCGAGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCTC GACACC NNNNNNN CNNNNNNNNNN  v12: (SEQ ID NO: 115)GGTGTCGAGAAGAGGAGAACAATATGCTAAATGTTGTTCTCGTCTCCTC GACACC NNNNNNN CNNNNNNNNNN  v13: (SEQ ID NO: 116)GGTGTCGAGAAGAGGAGAACANTAGGCTAAACGTTGTTTCTCGTCTCCT CGACACC NNNNNNN CNNNNNNNNNN 

The structure of V2 after folding is provided as FIG. 23D. And thecorresponding radRNAs were generated as follows:

(SEQ ID NO: 133) NNNNNNNNNNNNNNN C NNNTCCACCCTATGATATTGTTGTAAATCGTATAACAATATGATAAGGTGGG  (SEQ ID NO: 134) NNNNNNNNNNNNN CNNNNNNCACCCTATGATATTGTTGTAAATCGTATAAC AATATGATAAGGTG  (SEQ ID NO: 135)NNNNNNNNNNNNNN C NNNNNNCACCCTCTGCTCTTGTTGTAAATCGTATAA CAAGAGGAGAAGGTG (SEQ ID NO: 136) NNNNNNNNNNNNNNN C NNNTCCACCCTCTGCTCTTGTTGTAAATCGTATAACAAGAGGAGAAGGTGGG  (SEQ ID NO: 137) NNNNNNNNNNNNNN CNNNNNNCCACCTCTGCTCTTGTTGTAAATCGTATAA CAAGAGGAGAAGTGG  (SEQ ID NO: 138)NNNNNNNNNNNNN C NNNNNNNCCACCTCTGCTCTTGTTGTAAATCGTATAA CAAGAGGAGAAGTGG (SEQ ID NO: 139) NNNNNNNNNNNNNN C NNNNNNCACCCTCTGCTCTTGTTGCAAATCGGATAACAAGAGGAGAAGGTG  (SEQ ID NO: 140) NNNNNNNNNNNNNNN CNNNTCCACCCTCTGCTCTTGTTGCAAATCGGATAA CAAGAGGAGAAGGTGGG  (SEQ ID NO: 141)NNNNNNNNNNNNNN C NNNNNNCCACCTCTGCTCTTGTTGCAAATCGGATAA CAAGAGGAGAAGTGG NNNNNNNNNNNNN C NNNNNNNCCACCTCTGCTCTTGTTGCAAATCGGATAACAAGAGGAGAAGTGG (SEQ ID NO: 142) (SEQ ID NO: 143) NNNNNNNNNN CNNNNNNCCACAGCTCCTCTGCTCTTGTTGCAAATCGGATA ACAAGAGGAGAAGAGCTGTGG (SEQ ID NO: 144) NNNNNNNNNN C NNNNNNNCCACAGCTCCTCTGCTCTTGTTGTAAATCGTATAACAAGAGGAGAAGAGCTGTGG  (SEQ ID NO: 145) NNNNNNNNNN CNNNNNNNCCACAGCTCCTATGATATTGTTGTAAATCGTAT AACAATATGATAAGAGCTGTGG 

A schematic of the resulting adRNA and radRNA pairings to the targetmRNA is shown in FIG. 16C.

An alternative scaffold framework was also applied by Applicants usingtwo ADAR recruiting domains (black font) on either side of the targetingdomain while varying the position of the C mismatch in the targetingdomain (italicized Ns).

(SEQ ID NO: 146) TGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCAC NNNNNN NNNNNNNNNNNNNN GTGGAATAGTATAACAATATGCTAAATGTTGTTATA GTATCCCAC 

These non-limiting exemplary scaffolds provide a template for theengineering of adRNA and radRNA for particular targets and may beoptimized based on comparative efficacy studies carried out according tothe exemplary methods disclosed herein.

In some embodiments, the ADAR based editing system further comprisesADAR1, ADAR2, the E488Q and E100Q mutants each thereof, a fusion proteincomprising the catalytic domain of an ADAR and a domain which associateswith an RNA hairpin motif, a fusion protein comprising the catalyticdomain of an ADAR and a dead Cas9, or a fusion protein comprising thedouble stranded binding domain of an ADAR and an APOBEC. In furtherembodiments, the domain which associates with an RNA hairpin motif isselected from the group of an MS2 bacteriophage coat protein (MCP) andan N22 peptide. In some embodiments, the adRNA comprises one or more RNAhairpin motifs. In some embodiments, the one or more RNA hairpin motifsare selected from the group of an MS2 stem loop and a BoxB loop.

Not to be bound by theory, Applicants believe the double stranded RNAbinding motif from ADARs may bind to several double stranded RNAsequences and could thus have possible off target effects. To avoid sucheffects, Applicants contemplate the use of exogenous protein domains torecognize RNA hairpin motifs in the adRNA. Both ADAR1 and ADAR2 consistof RNA binding domains and a catalytic domain that catalyzes theconversion of adenosine to inosine. The catalytic domain can beuncoupled from the RNA binding domain. Our aim is to achieve highediting efficiency of the targeted adenosine while reducing off targeteffects and thus are exploring alternative RNA binding domains.Applicants have fused the catalytic domain of the ADAR1 or ADAR2 todifferent RNA binding domains such as the MCP, N22 or a dead CjCas9 (orother RNA targeting CRISPRs such as from SaCas9, CRISPR-Cas13 etc.).Upon the addition of appropriate guide RNAs (adRNAs), the fusionproteins are recruited to the target, further catalyzing an adenosine toinosine change. The dead CjCas9 (and other CRISPRs by extension) in thiscase basically serves as a RNA binding domain that can in turn betethered to effectors.

The domains are fused to the ADAR catalytic domain to generate ADARspecifically targeting the particular adRNA comprising the RNA hairpinmotifs. For example, Applicants have used a MS2 bacteriophage coatprotein (MCP) fused to either the catalytic domain of ADAR1 or ADAR2 andtheir respective mutants E488Q and E1008Q, while using a MS2 stem loopon the RNA to recruit the fusion protein (FIG. 23A). Analogous to thissystem, Applicants have also utilized a N22 peptide fused to thecatalytic domains of ADAR1 or ADAR2 (and their mutants) while making useof a boxB aptamer to recruit the fusion protein. Thus, one or two copiesof ADAR may be recruited based on the addition of single or dualhairpins (MS2/BoxB loops) (FIG. 23A). PP7 hairpins are also contemplatedfor use in the same manner.

A non-limiting framework sequence for the recruitment of MCP-basedfusion proteins, where the C mismatch may be varied within the targetingdomain, is provided herein below (with the lower case lettersrepresenting those linkers that help stabilize the underlined hairpins):

Single recruiting domain (underlined):

(SEQ ID NO: 98) NNNNNNNNNNNNNNNNNNNNggcc AACATGAGGATCACCCATGT CTGCAGggccTwo recruiting domains (underlined):

(SEQ ID NO: 99) a ACATGAGGATCACCCATGT cNNNNNNNNNNNNNNNNNNNNa ACATGAGGATCACCCATGT cAn analogous non-limiting framework sequence is provided for theN22-based fusion proteins:Single recruiting domain (underlined):

(SEQ ID NO: 100) NNNNNNNNNNNNNNNNNNNNgg gccctgaagaagggc ccTwo recruiting domains (underlined):

(SEQ ID NO: 101) gg GCCCTGAAGAAGGGC ccNNNNNNNNNNNNNNNNNNNNgg GCCCTGAAGAAGGGC cc

Another approach is to recruitment domains in the adRNA associated withCas9 and couple a dead Cas9 to the ADAR catalytic domain, thus,rendering the same effect of specific recruitment. A non-limitingframework sequence for the recruitment is provided for Cas9-based fusionproteins:

Psp dCas13a recruitment (mismatch position can be varied)

(SEQ ID NO: 147) CAACATTATCGGGGAGTTTTGACCTCCAAGGTGTTGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

Cj dCas9 recruitment (mismatch position can be varied)

(SEQ ID NO: 148) NNNNNNNNNNNNNNNNNNNNNNgttttagtccctgaaaagggactaaaataaagagtttgcgggactctgcggggttacaatcccctaaaaccgcttt tttt

APOBECs also have RNA editing function (FIG. 23B). Thus, they may beused in the alternative or in addition to the ADAR based editing system.For example, Applicants have created MCP/N22 peptide fusions withAPOBECs to engineer targeted C→T RNA editing. In addition, Applicantshave fused the double stranded RNA binding domains (dsRBD) of the ADAR2with APOBECs as a result of which the APOBECs can be recruited by theadRNA.

The addition of Nuclear Localization Signals (NLS) to the fusion proteincould help target nuclear RNA (i.e. pre-mRNA) while addition of NuclearExport Signals (NES) to the fusion protein could help target cytoplasmicRNA in any of the embodiments disclosed herein. This method is usefulwhen editing for splice site mutations, which result in incorrectprocessing of introns in the pre-mRNA and, thus, results in incorrectmRNA for translation. OTC deficiency is example where targeting pre-mRNAwith adRNA scaffolds can be useful, since the majority of aberrant OTCexpression comes from the splice site mutation resulting in a truncatedOTC protein. Further addition of RNA localization tags to the adRNA willenable targeting RNA in specific cellular compartments.

In further embodiments where the adRNA comprises one or more RNA hairpinmotifs, the one or more RNA hairpin motifs are stabilized by replacingA-U with G-C. In some embodiments, the adRNA is stabilized through theincorporation of one or more of 2′-O-methyl, 2′-O-methyl3′phosphorothioate, or 2′-O-methyl 3′thioPACE at either or both terminiof the adRNA.

More generally, can be appreciated that the RNA targeting domains ofadRNAs are designed such that they are complementary to the target mRNAwhile containing C mismatch at the position of the target adenosine. Therecruiting domains of the adRNA are constant. BY way of non-limitingexample:

Example target: OTC mRNA (mutation underlined)

(SEQ ID NO: 149) 5′-AAAGTCTCACAGACACCGCTC

GTTTGTAAAACTTTTCTTC-3′adRNA v2 (targeting domain length 20 bp, mismatch position after 6bases):

(SEQ ID NO: 149) 5′-AAAGTCTCACAGACACCGCTC

GTTTGTAAAACTTTTCTTC-3′ (SEQ ID NO: 150)5′-GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACT GTCTGTGGCGAGCCAAACA-3′adRNA v2 (targeting domain length 21 bp, mismatch position after 6bases):

(SEQ ID NO: 149) 5′-AAAGTCTCACAGACACCGCTC

GTTTGTAAAACTTTTCTTC-3′ (SEQ ID NO: 151)5′-GTGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACGTGTCTGTGGCGAGCCAAACA-3′radRNA v2 (targeting domain length 20 bp, mismatch position after 6bases):

(SEQ ID NO: 149) 3′-CTTCTTTTCAAAATGTTTG

CTCGCCACAGACACTCTGAAA-5′ (SEQ ID NO: 152)5′-AAGTTTTACAAACCGAGCGGCACCCTATGATATTGTTGTAAATCGT ATAACAATATGATAAGGTG-3′adRNA dual (targeting domain length 20 bp, mismatch position after 5, 14bases):

(SEQ ID NO: 149) 3′-CTTCTTTTCAAAATGTTTG

CTCGCCACAGACACTCTGAAA-5′ (SEQ ID NO: 153)5′-TGGAATAGTATAACAATATGCTAAATGTTGTTATAGTATCCCACCAAACCGAGCGGTGTCTGTGGTGGAATAGTATAACAATATGCTAAATGT TGTTATAGTATCCCAC-3′adRNA MS2 (targeting domain length 20 bp, mismatch position after 14bases)

(SEQ ID NO: 149) 3′-CTTCTTTTCAAAATGTTTG

CTCGCCACAGACACTCTGAAA-5′ (SEQ ID NO: 154)5′-CAAACCGAGCGGTGTCTGTGggccAACATGAGGATCACCCATGTCT GCAGggcc-3′adRNA MS2 dual (targeting domain length 20 bp, mismatch position after5, 14 bases)

(SEQ ID NO: 149) 5′-CTTCTTTTCAAAATGTTTG

CTCGCCACAGACACTCTGAAA-5′ (SEQ ID NO: 155)5′-aACATGAGGATCCCATGTcCAAACCGAGCGGTGTCTGTGaACATGA GGATCACCCATGTc-3′adRNA BoxB (targeting domain length 20 bp, mismatch position after 14bases)

(SEQ ID NO: 149) 3′-CTTCTTTTCAAAATGTTTG

CTCGCCACAGACACTCTGAAA-5′ (SEQ ID NO: 156)5′-CAAACCGAGCGGTGTCTGTGgggccctgaagaagggccc-3′adRNA BoxB dual (targeting domain length 20 bp, mismatch position after5, 14 bases)

(SEQ ID NO: 149) 3′-CTTCTTTTCAAAATGTTTG

CTCGCCACAGACACTCTGAAA-5′ (SEQ ID NO: 157)5′-ggGCCCTGAAGAAGGGCccCAAACCGAGCGGTGTCTGTGggGCCC TGAAGAAGGGCcc-3′

A coordinate or alternate approach to preventing off-target effects isto make use of endogenous ADAR. ADAR2 is highly expressed in tissuessuch as the brain, lung and spleen while ADAR1 is ubiquitously expressedwith general expression levels being higher than ADAR1. Thus, Applicantspropose two avenues in order to engineer RNA editing by endogenousADARs. First, ADAR1 expression can be stimulated by molecules such asinterferons, e.g., interferon α. Second, scaffolds may be engineeredspecifically for recruiting ADAR1 and are carrying out experiments withthe v1-v13 scaffolds as well as some chemically modified scaffoldsdisclosed herein above. Making use of the endogenous ADARs as opposed tooverexpression could help limit the off-target effects.

Recombinant Expression Systems and Vectors

Aspects of the disclosure relate to vectors and recombinant expressionsystems.

For example, some aspects relate to a vector encoding one or more tRNAhaving an anticodon sequence that recognizes a codon comprising a pointmutation in an RNA sequence encoding a protein, optionally wherein thepoint mutation results in a premature stop codon. In some embodiments,the point mutation results in a nonsense mutation having the DNAsequence TAA and the RNA sequence UAA. In some embodiments, the tRNA isan endogenous tRNA with a modified anticodon stem recognizing the codoncomprising the point mutation. In further embodiments, the tRNA ischarged with a serine. In some embodiments, the tRNA is an orthogonaltRNA charged with a non-canonical amino acid. In further embodiments,the vector further comprises a corresponding tRNA synthetase. In someembodiments, the corresponding synthetase is E. coli Glutaminyl-tRNAsynthetase. In some embodiments involving an orthogonal tRNA, thenon-canonical amino acid is pyrrolysine. In some embodiments, the vectorencodes two tRNA having an anticodon sequence that recognizes the codoncomprising the point mutation. In some embodiments, the vector is an AAVvector, optionally an AAV8 vector. In some embodiments, the protein isdystrophin.

Further aspects relate to a recombinant expression system comprising oneor more vectors encoding an ADAR based RNA editing system comprising oneor more forward guide RNAs for the ADAR (“adRNAs”) and one or morecorresponding reverse guide RNAs for the ADAR (“radRNAs”) to thesubject, wherein the ADAR based RNA editing system specifically edits apoint mutation in an RNA sequence encoding a protein. In someembodiments, the point mutation results in a nonsense mutation,optionally a premature stop codon, having the DNA sequence TAA and theRNA sequence UAA. In some embodiments, the ADAR based RNA editing systemconverts UAA to UIA and, optionally, further UIA to UM In someembodiments, the ADAR based RNA editing system converts UAA to UAI. Insome embodiments, the point mutation results in a splice site ormissense mutation having the DNA sequence CAG and the RNA sequence CAG.In some embodiments, the ADAR based RNA editing system converts CAG toCIG. In further embodiments, the one or more vector further encodes atRNA that targets an amber codon. In some embodiments, the ADAR basedediting system further comprises ADAR1, ADAR2, the E488Q and E100Qmutants each thereof, a fusion protein comprising the catalytic domainof an ADAR and a domain which associates with an RNA hairpin motif, afusion protein comprising the catalytic domain of an ADAR and a deadCas9, or a fusion protein comprising the double stranded binding domainof an ADAR and an APOBEC. In further embodiments, the domain whichassociates with an RNA hairpin motif is selected from the group of anMS2 bacteriophage coat protein (MCP) and an N22 peptide. In someembodiments, the adRNA comprises one or more RNA hairpin motifs. In someembodiments, the one or more RNA hairpin motifs are selected from thegroup of an MS2 stem loop and a BoxB loop and/or are stabilized byreplacing A-U with G-C. In some embodiments, the adRNA is stabilizedthrough the incorporation of one or more of 2′-O-methyl, 2′-O-methyl3′phosphorothioate, or 2′-O-methyl 3′thioPACE at either or both terminiof the adRNA.

In general methods of packaging genetic material such as RNA into one ormore vectors is well known in the art. For example, the genetic materialmay be packaged using a packaging vector and cell lines and introducedvia traditional recombinant methods.

In some embodiments, the packaging vector may include, but is notlimited to retroviral vector, lentiviral vector, adenoviral vector, andadeno-associated viral vector (optionally AAV8). The packaging vectorcontains elements and sequences that facilitate the delivery of geneticmaterials into cells. For example, the retroviral constructs arepackaging plasmids comprising at least one retroviral helper DNAsequence derived from a replication-incompetent retroviral genomeencoding in trans all virion proteins required to package a replicationincompetent retroviral vector, and for producing virion proteins capableof packaging the replication-incompetent retroviral vector at hightiter, without the production of replication-competent helper virus. Theretroviral DNA sequence lacks the region encoding the native enhancerand/or promoter of the viral 5′ LTR of the virus, and lacks both the psifunction sequence responsible for packaging helper genome and the 3′LTR,but encodes a foreign polyadenylation site, for example the SV40polyadenylation site, and a foreign enhancer and/or promoter whichdirects efficient transcription in a cell type where virus production isdesired. The retrovirus is a leukemia virus such as a Moloney MurineLeukemia Virus (MMLV), the Human Immunodeficiency Virus (HIV), or theGibbon Ape Leukemia virus (GALV). The foreign enhancer and promoter maybe the human cytomegalovirus (HCMV) immediate early (IE) enhancer andpromoter, the enhancer and promoter (U3 region) of the Moloney MurineSarcoma Virus (MMSV), the U3 region of Rous Sarcoma Virus (RSV), the U3region of Spleen Focus Forming Virus (SFFV), or the HCMV IE enhancerjoined to the native Moloney Murine Leukemia Virus (MMLV) promoter.

The retroviral packaging plasmid may consist of two retroviral helperDNA sequences encoded by plasmid based expression vectors, for examplewhere a first helper sequence contains a cDNA encoding the gag and polproteins of ecotropic MMLV or GALV and a second helper sequence containsa cDNA encoding the env protein. The Env gene, which determines the hostrange, may be derived from the genes encoding xenotropic, amphotropic,ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virusenv proteins, or the Gibbon Ape Leukemia Virus (GALV env protein, theHuman Immunodeficiency Virus env (gp160) protein, the VesicularStomatitus Virus (VSV) G protein, the Human T cell leukemia (HTLV) typeI and II env gene products, chimeric envelope gene derived fromcombinations of one or more of the aforementioned env genes or chimericenvelope genes encoding the cytoplasmic and transmembrane of theaforementioned env gene products and a monoclonal antibody directedagainst a specific surface molecule on a desired target cell. Similarvector based systems may employ other vectors such as sleeping beautyvectors or transposon elements.

The resulting packaged expression systems may then be introduced via anappropriate route of administration, discussed in detail with respect tothe method aspects disclosed herein.

Compositions

Further aspects relate to a composition comprising any one or more ofthe vectors disclosed herein. In some embodiments, the compositionfurther comprises an effective amount of an interferon to enhanceendogenous ADAR1 expression. In still further embodiments, theinterferon is interferon α.

Briefly, pharmaceutical compositions of the present disclosure includingbut not limited to any one of the claimed compositions may comprise atarget cell population as described herein, in combination with one ormore pharmaceutically or physiologically acceptable carriers, diluentsor excipients.

Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of thedisclosure. Those skilled in the art will know of other suitablecarriers for binding antibodies, or will be able to ascertain such,using routine experimentation.

Such compositions may also comprise buffers such as neutral bufferedsaline, phosphate buffered saline and the like; carbohydrates such asglucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptidesor amino acids such as glycine; antioxidants; chelating agents such asEDTA or glutathione; adjuvants (e.g., aluminum hydroxide); andpreservatives. Compositions of the present disclosure may be formulatedfor oral, intravenous, topical, enteral, and/or parenteraladministration. In certain embodiments, the compositions of the presentdisclosure are formulated for intravenous administration.

Administration of the compositions can be effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are known to those of skill in the art and will vary withthe composition used for therapy, the purpose of the therapy and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician. Suitable dosage formulations and methods of administering theagents are known in the art. In a further aspect, the cells andcomposition of the disclosure can be administered in combination withother treatments.

The vectors, recombinant expression systems, and/or compositions areadministered to the host using methods known in the art. Thisadministration of the compositions of the disclosure can be done togenerate an animal model of the desired disease, disorder, or conditionfor experimental and screening assays.

Briefly, pharmaceutical compositions of the present disclosure includingbut not limited to any one of the claimed compositions may comprise oneor more vectors or recombinant expression systems as described herein,in combination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present disclosure may be formulated for oral, intravenous, topical,enteral, and/or parenteral administration. In certain embodiments, thecompositions of the present disclosure are formulated for intravenousadministration.

Pharmaceutical compositions of the present disclosure may beadministered in a manner appropriate to the disease, disorder, orcondition to be treated or prevented. The quantity and frequency ofadministration will be determined by such factors as the condition ofthe patient, and the type and severity of the patient's disease,although appropriate dosages may be determined by clinical trials.

Methods of Restoring Protein Expression

Aspects of the disclosure relate to methods of restoring proteinexpression.

For example, some aspects of the disclosure relate to a method forrestoring expression of a protein comprising a point mutation in an RNAsequence encoding the protein in a subject in need thereof comprisingadministering a vector encoding one or more tRNA having an anticodonsequence that recognizes a codon comprising the point mutation to thesubject, optionally wherein the point mutation results in a prematurestop codon. In some embodiments, the point mutation results in anonsense mutation having the DNA sequence TAA and the RNA sequence UAA.In some embodiments, the tRNA is an endogenous tRNA with a modifiedanticodon stem recognizing the codon comprising the point mutation. Infurther embodiments, the tRNA is charged with a serine. In someembodiments, the tRNA is an orthogonal tRNA charged with a non-canonicalamino acid. In further embodiments, the vector further comprises acorresponding tRNA synthetase. In some embodiments, the correspondingsynthetase is E. coli Glutaminyl-tRNA synthetase. In some embodimentsinvolving an orthogonal tRNA, the non-canonical amino acid ispyrrolysine. In further embodiments, the pyrrolysine is introduced inthe diet of the subject. In some embodiments, the vector encodes twotRNA having an anticodon sequence that recognizes the codon comprisingthe point mutation. In some embodiments, the protein is dystrophin.

Other aspects relate to a recombinant expression system comprising oneor more vectors encoding an ADAR based RNA editing system comprising oneor more forward guide RNAs for the ADAR (“adRNAs”) and one or morecorresponding reverse guide RNAs for the ADAR (“radRNAs”) to thesubject, wherein the ADAR based RNA editing system specifically edits apoint mutation in an RNA sequence encoding a protein. In someembodiments, the point mutation results in a nonsense mutation,optionally a premature stop codon, having the DNA sequence TAA and theRNA sequence UAA. In some embodiments, the ADAR based RNA editing systemconverts UAA to UIA and, optionally, further UIA to UM In someembodiments, the ADAR based RNA editing system converts UAA to UAI. Insome embodiments, optionally those involving nonsense or missensemutations, the RNA targeted in mRNA. In further embodiments, the one ormore vector further encodes a tRNA that targets an amber codon. In someembodiments, the protein is dystrophin. In some embodiments, the pointmutation results in a splice site or missense mutation having the DNAsequence CAG and the RNA sequence CAG. In some embodiments, the ADARbased RNA editing system converts CAG to CIG. In some embodiments,optionally those involving splice site mutations, the RNA targeted ispre-mRNA. In some embodiments, the ADAR based editing system furthercomprises ADAR1, ADAR2, the E488Q and E100Q mutants each thereof, afusion protein comprising the catalytic domain of an ADAR and a domainwhich associates with an RNA hairpin motif, a fusion protein comprisingthe catalytic domain of an ADAR and a dead Cas9, or a fusion proteincomprising the double stranded binding domain of an ADAR and an APOBEC.In further embodiments, the domain which associates with an RNA hairpinmotif is selected from the group of an MS2 bacteriophage coat protein(MCP) and an N22 peptide. In some embodiments, the adRNA comprises oneor more RNA hairpin motifs. In some embodiments, the one or more RNAhairpin motifs are selected from the group of an MS2 stem loop and aBoxB loop and/or are stabilized by replacing A-U with G-C. In someembodiments, the adRNA is stabilized through the incorporation of one ormore of 2′-O-methyl, 2′-O-methyl 3′phosphorothioate, or 2′-O-methyl3′thioPACE at either or both termini of the adRNA.

In either case, the assessment of whether protein expression is“restored” is achieved through any means of protein quantification whencompared to a baseline. The baseline may optionally be calculated basedon a prior level in the subject or as the normal level in thepopulation, adjusted for the subject's age, ethnicity, and otherrelevant demographic information. Techniques of quantifying proteinexpression are well known in the art and may, optionally, utilize acontrol or a threshold value for comparison to the baseline value.Methods known in the art for such studies include but are not limited toqRTPCR, ELISA, Western blot, protein immunostaining, spectroscopy and/orspectrometry based methods, and other assays typically conducted todetermine the amount of protein expression in a sample from the subject.Alternatively, the “restoration” effect may be determined based on aclinical outcome. For example, aberrant dystrophin levels are linked tomuscular dystrophy symptoms. Thus, the restoration of expression may beoutwardly determined based on clinical signals such as a reduction orreversal of these symptoms. For dystrophin, improvement in musclestrength can be one such indicator. Thus, physicians may carry outstrength measurements to determine outcome. Another example is ornithinetranscarbamylase (OTC); aberrant OTC levels are a result of a rareX-linked genetic disorder resulting in excessive accumulation of ammoniain the blood (due to nitrogen accumulation). Thus, a relevant clinicaloutcome would be a decrease in ammonia in a biological sample, such asblood or urine. Similarly, clinical signals associated with andexpression of proteins downstream of the protein of interest may berelevant indicators of “restoration” where the protein of interest isinvolved in a particular pathway.

Methods of Treatment

Point mutations are implicated in a number of diseases, disorders, andconditions. Non-limiting examples are provided in Table 1 below.

TABLE 1 Protein/Disease, Disorder, or Condition Associated PointMutation G to A point mutations or premature stop codonsDihydropyrimidine dehydrogenase deficiency NM_000110.3(DPYD):c.1905+1G > A Noonan syndrome NM_005633.3(SOS1): c.2536G > A(p.Glu846Lys) Lynch syndrome NM_000251.2(MSH2): c.212−1G > ABreast-ovarian cancer, familial 1 NM_007294.3(BRCA1): c.963G > A(p.Trp321Ter) Cystic fibrosis NM_000492.3(CFTR): c.57G > A (p.Trp19Ter)Anemia, due to G6PD deficiency NM_000402.4(G6PD): c.292G > A(p.Val98Met) AVPR2 Nephrogenic diabetes insipidus, X-linkedNM_000054.4(AVPR2): c.878G > A (p.Trp293Ter) FANCC Fanconi anemia,complementation group C NM_000054.4(AVPR2): c.878G > A (p.Trp293Ter)FANCC Fanconi anemia, complementation group C NM_000136.2(FANCC):c.1517G > A (p.Trp506Ter) IL2RG X-linked severe combinedNM_000206.2(IL2RG): c.710G > A (p.Trp237Ter) immunodeficiency F8Hereditary factor VIII deficiency disease NM_000132.3(F8): c.3144G > A(p.Trp1048Ter) LDLR Familial hypercholesterolemia NM_000527.4(LDLR):c.1449G > A (p.Trp483Ter) CBS Homocystinuria due to CBS deficiencyNM_000071.2(CBS): c.162G > A (p.Trp54Ter) HBB betaThalassemiaNM_000518.4(HBB): c.114G > A (p.Trp38Ter) ALDOB Hereditary fructosuriaNM_000035.3(ALDOB): c.888G > A (p.Trp296Ter) DMD Duchenne musculardystrophy NM_004006.2(DMD): c.3747G > A (p.Trp1249Ter) SMAD4 Juvenilepolyposis syndrome NM_005359.5(SMAD4): c.906G > A (p.Trp302Ter) BRCA2Familial cancer of breast|Breast-ovarian NM_000059.3(BRCA2): c.582G > A(p.Trp194Ter) cancer, familial 2 GRIN2A Epilepsy, focal, with speechdisorder and NM_000833.4(GRIN2A): c.3813G > A (p.Trp1271Ter) with orwithout mental retardation SCN9A Indifference to pain, congenital,autosomal NM_002977.3(SCN9A): c.2691G > A (p.Trp897Ter) recessive TARDBPAmyotrophic lateral sclerosis type 10 NM_007375.3(TARDBP): c.943G > A(p.Ala315Thr) CFTR Cystic fibrosis|Hereditary pancreatitis|notNM_000492.3(CFTR): c.3846G > A (p.Trp1282Ter) provided|atalurenresponse - Efficacy UBE3A Angelman syndrome NM_130838.1(UBE3A):c.2304G > A (p.Trp768Ter) SMPD1 Niemann-Pick disease, type ANM_000543.4(SMPD1): c.168G > A (p.Trp56Ter) USH2A Usher syndrome, type2A NM_206933.2(USH2A): c.9390G > A (p.Trp3130Ter) MEN1 Hereditarycancer-predisposing syndrome NM_130799.2(MEN1): c.1269G > A(p.Trp423Ter) C8orf37 Retinitis pigmentosa 64 NM_177965.3(C8orf37):c.555G > A (p.Trp185Ter) MLH1 Lynch syndrome NM_000249.3(MLH1):c.1998G > A (p.Trp666Ter) TSC2 Tuberous sclerosis 2|Tuberous sclerosisNM_000548.4(TSC2): c.2108G > A (p.Trp703Ter) syndrome 46 NF1Neurofibromatosis, type 1 NM_000267.3(NF1): c.7044G > A (p.Trp2348Ter)MSH6 Lynch syndrome NM_000179.2(MSH6): c.3020G > A (p.Trp1007Ter) SMN1Spinal muscular atrophy, type II|Kugelberg- NM_000344.3(SMN1): c.305G >A (p.Trp102Ter) Welander disease SH3TC2 Charcot-Marie-Tooth disease,type 4C NM_024577.3(SH3TC2): c.920G > A (p.Trp307Ter) DNAH5 Primaryciliary dyskinesia NM_001369.2(DNAH5): c.8465G > A (p.Trp2822Ter) MECP2Rett syndrome NM_004992.3(MECP2): c.311G > A (p.Trp104Ter) ADGRV1 Ushersyndrome, type 2C NM_032119.3(ADGRV1): c.7406G > A (p.Trp2469Ter) AHI1Joubert syndrome 3 NM_017651.4(AHI1): c.2174G > A (p.Trp725Ter) PRKNParkinson disease 2 NM_004562.2(PRKN): c.1358G > A (p.Trp453Ter) COL3A1Ehlers-Danlos syndrome, type 4 NM_000090.3(COL3A1): c.3833G > A(p.Trp1278Ter) BRCA1 Familial cancer of breast|Breast-ovarianNM_007294.3(BRCA1): c.5511G > A (p.Trp1837Ter) cancer, familial 1 MYBPC3Primary familial hypertrophic NM_000256.3(MYBPC3): c.3293G > Acardiomyopathy (p.Trp1098Ter) APC Familial adenomatous polyposis 1NM_000038.5(APC): c.1262G > A (p.Trp421Ter) BMPR2 Primary pulmonaryhypertension NM_001204.6(BMPR2): c.893G > A (p.W298*) T to C pointmutations Wilson disease NM_000053.3(ATP7B): c.3443T > C (p.Ile1148Thr)Leukodystrophy, hypomyelinating, 2 NM_020435.3(GJC2): c.857T > C(p.Met286Thr) Alport syndrome, X-linked recessive NM_000495.4(COL4 A5):c.438+2T > C Leigh disease NC_012920.1: m.9478T > C Gaucher disease,type 1 NM_001005741.2(GBA): c.751T > C (p.Tyr251His) Renal dysplasia,retinal pigmentary dystrophy, NM_014714.3(IFT140): c.4078T > C(p.Cys1360Arg) cerebellar ataxia and skeletal dysplasia Marfan syndromeNM_000138.4(FBN1): c.3793T > C (p.Cys1265Arg) Deficiency ofUDPglucose-hexose-1-phosphate NM_000155.3(GALT): c.482T > C(p.Leu161Pro) uridylyltransferase Familial hypercholesterolemiaNM_000527.4(LDLR): c.694+2T > C Episodic pain syndrome, familial, 3NM_001287223.1(SCN11A): c.1142T > C (p.Ile381Thr) Navajoneurohepatopathy NM_002437.4(MPV17): c.186+2T > C Congenital musculardystrophy, LMNA-related NM_170707.3(LMNA): c.1139T > C (p.Leu380Ser)Hereditary factor VIII deficiency disease NM_000132.3(F8): c.5372T > C(p.Met1791Thr) Insulin-dependent diabetes mellitus secretoryNM_014009.3(FOXP3): c.970T > C (p.Phe324Leu) diarrhea syndromeHereditary factor IX deficiency disease NM_000133.3(F9): c.1328T > C(p.Ile443Thr) Familial cancer of breast, Breast-ovarian cancer,NM_000059.3(BRCA2): c.316+2T > C familial 2, Hereditary cancerpredisposing syndrome Cardiac arrhythmia NM_000238.3(KCNH2): c.1945+6T >C Tangier disease NM_005502.3(ABCA1): c.4429T > C (p.Cys1477Arg) Dilatedcardiomyopathy 1AA NM_001103.3(ACTN2): c.683T > C (p.Met228Thr) Mentalretardation 3, X-linked NM_005334.2(HCFC1): c.−970T > C Limb-girdlemuscular dystrophy, type 2B NM_003494.3(DYSF): c.1284+2T > C Maculardystrophy, vitelliform, 5 NM_016247.3(IMPG2): c.370T > C (p.Phe124Leu)Retinitis pigmentosa NM_000322.4(PRPH2): c.736T > C (p.Trp246Arg)

Further non-limiting examples include Ornithine TranscarbamylaseDeficiency, Nougaret night blindness, Usher syndrome, AtrialFibrillation, Duchenne Muscular Dystrophy, Wilson disease, hereditarytyrosinemia, and some cancers carrying a A→G mutation in genes such asB-catenin.

Thus, aspects of this disclosure relate to the treatment of certaindiseases, disorders, and conditions involving point mutations.

For example, some method aspects relate to a treating a disease,disorder, or condition characterized by the presence of a point mutationin an RNA sequence encoding a protein associated with the disease,disorder, or condition in a subject in need thereof comprisingadministering a vector encoding one or more tRNA having an anticodonsequence that recognizes a codon comprising the point mutation to thesubject, optionally wherein the point mutation results in a prematurestop codon. In some embodiments, the point mutation results in anonsense mutation having the DNA sequence TAA and the RNA sequence UAA.In some embodiments, the tRNA is an endogenous tRNA with a modifiedanticodon stem recognizing the codon comprising the point mutation. Infurther embodiments, the tRNA is charged with a serine. In someembodiments, the tRNA is an orthogonal tRNA charged with a non-canonicalamino acid. In further embodiments, the vector further comprises acorresponding tRNA synthetase. In some embodiments, the correspondingsynthetase is E. coli Glutaminyl-tRNA synthetase. In some embodimentsinvolving an orthogonal tRNA, the non-canonical amino acid ispyrrolysine. In further embodiments, the pyrrolysine is introduced inthe diet of the subject. In some embodiments, the vector encodes twotRNA having an anticodon sequence that recognizes the codon comprisingthe point mutation. In some embodiments, the disease, disorder, orcondition is selected from the group consisting of the diseases,disorders, and conditions listed in Table 1, optionally characterized bythe presence of a nonsense mutation and/or a premature stop codon. Insome embodiments, the protein is dystrophin. In further embodiments, thedisease, disorder, or condition is muscular dystrophy. In still furtherembodiments, the disease disorder or condition is Duchenne musculardystrophy.

Additional method aspects relate to a method of treating a disease,disorder, or condition by the presence of a point mutation in an RNAsequence encoding a protein associated with the disease, disorder, orcondition in a subject in need thereof comprising administering one ormore vectors encoding an ADAR based RNA editing system comprising one ormore forward guide RNAs for the ADAR (“adRNAs”) and one or morecorresponding reverse guide RNAs for the ADAR (“radRNAs”) to thesubject, wherein the ADAR based RNA editing system specifically editsthe point mutation. In some embodiments, the point mutation results in anonsense mutation, optionally a premature stop codon, having the DNAsequence TAA and the RNA sequence UAA. In some embodiments, the ADARbased RNA editing system converts UAA to UIA and, optionally, furtherUIA to UII. In some embodiments, the ADAR based RNA editing systemconverts UAA to UAI. In some embodiments, optionally those involvingnonsense or missense mutations, the RNA targeted in mRNA. In furtherembodiments, the one or more vector further encodes a tRNA that targetsan amber codon. In some embodiments, the protein is dystrophin. In someembodiments, the point mutation results in a splice site or missensemutation having the DNA sequence CAG and the RNA sequence CAG. In someembodiments, the ADAR based RNA editing system converts CAG to CIG. Insome embodiments, optionally those involving splice site mutations, theRNA targeted is pre-mRNA. In some embodiments, the ADAR based editingsystem further comprises ADAR1, ADAR2, the E488Q and E100Q mutants eachthereof, a fusion protein comprising the catalytic domain of an ADAR anda domain which associates with an RNA hairpin motif, a fusion proteincomprising the catalytic domain of an ADAR and a dead Cas9, or a fusionprotein comprising the double stranded binding domain of an ADAR and anAPOBEC. In further embodiments, the domain which associates with an RNAhairpin motif is selected from the group of an MS2 bacteriophage coatprotein (MCP) and an N22 peptide. In some embodiments, the methodfurther comprises administering an effective amount of an interferon toenhance endogenous ADAR1 expression. In still further embodiments, theinterferon is interferon α. In some embodiments, the adRNA comprises oneor more RNA hairpin motifs. In some embodiments, the one or more RNAhairpin motifs are selected from the group of an MS2 stem loop and aBoxB loop and/or are stabilized by replacing A-U with G-C. In someembodiments, the adRNA is stabilized through the incorporation of one ormore of 2′-O-methyl, 2′-O-methyl 3′phosphorothioate, or 2′-O-methyl3′thioPACE at either or both termini of the adRNA. In some embodiments,the disease, disorder, or condition is selected from the groupconsisting of the diseases, disorders, and conditions listed in Table 1.In further embodiments, the protein is dystrophin and the disease,disorder, or condition is muscular dystrophy. In still furtherembodiments, the disease disorder or condition is Duchenne musculardystrophy.

An ordinary skilled artisan will appreciate that the doses and route ofadministration employed in these methods may vary based on the subjectand the disease, disorder, or condition to be treated. Based onknowledge in the art such suitable doses and routes may be selectedbased on the subject's age, ethnicity, and other relevant demographicfactors.

Kits

In one particular aspect, the present disclosure provides kits forperforming any of the methods disclosed herein as well as instructionsfor carrying out the methods of the present disclosure and/oradministering the vectors, recombinant expression systems, andcompositions disclosed herein.

The kit can also comprise agents necessary for the preservation of thosecomponents comprised therein, e.g., a buffering agent, a preservative ora protein-stabilizing agent. The kit can further comprise componentsnecessary for detecting the detectable-label, e.g., an enzyme or asubstrate. The kit can also contain a control sample or a series ofcontrol samples, which can be assayed and compared to the test sample.Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package, alongwith instructions for interpreting the results of the assays performedusing the kit. The kits of the present disclosure may contain a writtenproduct on or in the kit container. The written product describes how touse the reagents contained in the kit.

As amenable, these suggested kit components can be packaged in a mannercustomary for use by those of skill in the art. For example, thesesuggested kit components may be provided in solution or as a liquiddispersion or the like.

EXAMPLES

The following examples are non-limiting and illustrative of procedureswhich can be used in various instances in carrying the disclosure intoeffect. Additionally, all reference disclosed herein are incorporated byreference in their entirety.

Example 1—Design of tRNA Constructs

The tRNA constructs were designed along the lines of the schematics inFIG. 1 to recognize the nonsense mutation TAA. Both modified endogenousand orthogonal tRNA were generated. The constructs were validated invitro using a GFP harboring nonsense mutation. It was determined thattwo copies of the tRNA should be include in each AAV vector for bothmodified endogenous and orthogonal tRNAs. MbPyl synthetase was selectedfor use with the orthogonal tRNA. The AAV vectors were generatedcomprising the tRNA and GFP (as well as the synthetase, where orthogonaltRNA was used). The sequences used in these constructs are provided inthe Sequence Listing above.

Example 2—Restoration of Full Length Dystrophin in Mdx Mice

The anticodon stem of the human serine tRNA is modified such that itrecognizes the nonsense codon (TAA). No endogenous tRNA can recognize astop codon and translation terminates when the ribosome reaches a stopcodon. Mdx mice bear a nonsense mutation (TAA) in the gene coding fordystrophin as a result of which they lack full length dystrophinexpression. AAVs are used to deliver two copies of the modified tRNAsinto the mouse muscle which in turn allows for the stop codonread-through enabling the expression of full length dystrophin.

The calf muscles of mdx mice were injected with 1E12 particles of AAV8carrying 2 copies of the modified serine tRNA and a GFP gene. These micewere then sacrificed after a month and the calf muscles were harvested.The muscles were sectioned and stained with an antibody againstdystrophin. A clear restoration of dystrophin expression was noticed. Inaddition, the muscle morphology improved too.

Applicants have, thus, demonstrated activity of our vectors in vitrousing a GFP gene harboring a stop codon. In addition Applicants havedemonstrated restoration of dystrophin expression in mdx mouse muscles.Within a span of one month after injecting the mdx mice with AAVscarrying two copies of the serine tRNA, Applicants observed restorationof dystrophin expression in the calf muscle via Immunohistochemistry.Applicants also noted an improved muscle morphology.

This experiment is repeated with the orthogonal tRNA, introducing thepyrrolysine through the mouse feed, and is again replicated with bothtRNAs in a larger population of mice.

Example 3—Diet Regulable Production of Therapeutic Proteins

Applicants aim at achieving on-demand, in vivo production oftherapeutics such as (i) insulin; (ii) neutralizing antibodies forviruses (e.g. HIV, HCV, HPV, influenza) and bacteria (e.g. staph aureus;drug resistant strains) by the skeletal muscle.

The desired transgenes are delivered to the muscle via AAVs (orlentiviruses) along with an orthogonal tRNA/tRNA synthetase pair. Thesetransgenes contain a premature termination codon (stop codon) that willprevent the full length protein from being expressed. For an on demandsynthesis of the therapeutics, an appropriate unnatural amino acid isconsumed in the diet, which in turn is incorporated by the orthogonaltRNA/tRNA synthetase at the premature termination site, enablingsynthesis of the desired therapeutics.

Example 4—ADAR2 Based RNA Editing

Applicants suspected that ADAR2 (adenosine deaminase that acts on RNA)to correct mutations at the mRNA level. Applicants used Adeno-AssociatedViruses to deliver the ADAR2 and a adRNA (forward ADAR2 guide) or radRNA(reverse ADAR2 guide) that direct the enzyme to the mutation in anattempt to restore the expression of dystrophin in the mdx mouse modelsof DMD, by editing the nonsense mutation. Applicants also applied thistechnology to the mouse model of the metabolic disorder OrnithineTranscarbamylase (OTC) deficiency.

As compared to nucleases, ADARs make site specific Adenosine to Inosine(A→I) changes in the mRNA with Inosine being read as a Guanosine (G)during translation and are thereby safe from creating permanent offtarget effects. Also, since they make edits at the mRNA level, thealtered proteins are expressed only transiently. The use of nucleases inadult OTC-deficient mice led to large deletions that proved to be lethalto the animals. The use of ADARs might circumvent this problem. Thiscould be a readily translatable solution for several disorderscharacterized by point mutations. In addition, the origin of the ADAR2is human, thereby minimizing the immune response generated by the bodyagainst it. Applicants also combine the idea of tRNA suppression withADAR2 based RNA editing. In addition, Applicants designed hairpin loops(3′ overlap) and toe-holds (5′ overlap) that help improve thespecificity of the adRNA/radRNA. Applicants also go on to optimize thelengths of the adRNA for efficient A→I editing as well as the ADAR2recruiting domain of the adRNA/radRNA.

Existing studies have made use of nucleases such as Cas9 to delete themutated region of the Dystrophin/OTC genes and replaced it with afunctional copy, for the treatment of DMD or OTC deficiency caused by apoint mutation. For DMD, existing therapies include the use ofcorticosteroids that delay the symptoms of the disorder. Otherstrategies include the premature stop codon read-through by making useof drugs such as Ataluren or Gentamycin. Another strategy is that ofexon skipping which results in a truncated protein, however able toreduce the severity of the DMD phenotype. Another approach is thedelivery of a u-dystrophin gene. Clinical trials for OTC deficiency havebeen attempted making use of adenoviral vectors to deliver OTC cDNA inpatients. Other avenues for treatment include use of sodiumphenylbutyrate which helps increase the waste nitrogen excretion.

The use of ADAR2 as an engineered RNA editing enzyme has beendemonstrated only in vitro.

Applicants utilized adRNAs and radRNAs comprised of two domains, onecomplementary to the target sequence and the other an ADAR2 recruitingdomain. Applicants utilized AAVs to deliver these adRNAs/radRNAs alongwith the ADAR2 enzyme. Mdx mice bear a nonsense mutation (TAA) in thegene coding for dystrophin. Applicants packaged two copies of theadRNAs/radRNAs or a combination of adRNA/radRNA+tRNA along with theADAR2 enzyme into the AAV and deliver it into the Tibialis Anterior (TA)muscle. Applicants utilized three alternative strategies to restoredystrophin expression:

(1) adRNAs/radRNAs that can edit both the adenosines in the ‘TAA’ toinosines;

(2) a sequential approach whereby the first adRNA/radRNA convertsTAA→TGA and the next adRNA/radRNA converts it to TGG, restoringexpression; and

(3) a combination of adRNAs/radRNAs and a tRNA whereby the adRNA/radRNAconverts the TAA into TAG and the tRNA suppression of the amber codon(TAG) restores dystrophin expression.

Applicants also delivered two copies of the adRNA targeting the OTC G→Amutation in spf-ash mice along with the ADAR2 to the liver viaretro-orbital injections.

The system works by editing an Adenosine to Inosine which is read as aGuanosine during translation. This can be used to correct pointmutations as well as restore expression by editing premature stopcodons. In FIG. 6: A. An Amber stop codon can be converted to atryptophan codon by a single edit. B. Ochre stop codon—both edits madein a single step. C. Ochre stop codon—Sequential editing. D. Ochre stopcodon—ADAR2 editing in combination with suppressor tRNA.

The following 10 steps represent the workflow to test these constructs:

-   -   1. Design and clone ADAR2 constructs—adRNA and radRNA.    -   2. In vitro validation of constructs using a GFP harboring a        nonsense mutation.    -   3. Modification of constructs—decision to clone two copies of        the adRNA/radRNA. Creation of vectors harboring one copy of a        adRNA/radRNA and a copy of a serine suppressor tRNA.    -   4. Generation of AAV8 vectors carrying ADAR2 and adRNAs/radRNAs        or suppressor tRNAs.    -   6. TA/Gastrocnemius injections of mdx mice—1E12 particles of        AAV8 carrying the ADAR2 and with adRNAs/radRNAs or suppressor        tRNA were injected.    -   7. The mice were sacrificed 6 weeks after injections and the        TA/gastrocnemius were harvested. Immunohistochemistry performed        to detect dystrophin. Some evidence of restoration of        dystrophin.    -   8. qPCR, Western blots and NGS were carried out.    -   9. Vectors were optimized to improve efficiency. adRNA lengths        varied, location of the edit varied.    -   10. Steps 4-8 repeated with optimized vectors.

Applicants designed adRNA and radRNAs against a premature stop codon inGFP and demonstrated robust restoration of expression (FIG. 5). For theochre stop codon (TAA), two A→G edits are needed to restore expression.Applicants constructed a single ad/radRNA targeting both As or a twoad/radRNAs that target a single A in a sequential manner. Applicantsalso constructed an adRNA/radRNA+suppressor tRNA vector combining RNAediting with tRNA suppression.

In vitro RNA editing showed robust restoration of GFP expression afterwhich AAVs bearing the ADAR2 and adRNA/radRNAs were generated to targetthe nonsense mutation in dystrophin in mdx mice.

The Tibialis Anterior (TA) or gastrocnemius muscles of mdx mice wereinjected with 1E12 particles of AAV8 carrying ADAR2 and two copies ofthe adRNA/radRNA or one copy of the adRNA/radRNA and a suppressor tRNA.These mice were sacrificed after 6 weeks and the appropriate muscleswere harvested. The muscles were sectioned and stained with an antibodyagainst dystrophin. Partial restoration of dystrophin expression wasnoticed.

In general, Applicants noticed a fractional restoration of dystrophinexpression via Immunostaining. However, western blots and NGS did notshow any evidence of editing/restoration of dystrophin expression.

Potential applications of the system include targeting point mutationsfor the treatment of disorders such as but not restricted to DMD, OTCdeficiency, Wilson's disease and hereditary tyrosinemia type 1. It couldalso be used to create alternate start codons, enabling the co-existenceof a protein and its N-terminal truncated form.

Example 5—ADAR Editing in Mouse Models

Genome engineering methodologies coupled with rapidly advancingsynthetic biology toolsets are enabling powerful capabilities to perturbgenomes for deciphering function, programming novel function, andrepairing aberrant function. In particular, programmable DNA nucleases,such as meganucleases, zinc finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TALENs), and CRISPR-Cas, have beenwidely used to engineer genomes across a range of organisms. Their usein gene therapy however poses at least three major challenges: one, theefficiency of homologous recombination versus non-homologous end joiningis typically low, particularly in post-mitotic cells that comprise thevast majority of the adult body; two, an active nuclease always posesthe threat of introducing permanent off-target mutations, thuspresenting formidable challenges in both engineering exquisite nucleasespecificity without compromising activity, as well as ensuring tightregulation of the nuclease dose and duration in target cells; and three,prevalent programmable nucleases are of prokaryotic origin or beardomains that are of non-human origin raising a significant risk ofimmunogenicity in in vivo therapeutic applications. The recent advent ofbase editing approaches is opening an exciting alternative strategy forgene targeting, but demonstrated approaches rely on CRISPR-Cas systemsthat are of prokaryotic origin. Thus for genomic mutations that lead toalteration in protein function, such as in disease causing genemutations, approaches to instead directly target RNA would be highlydesirable. Leveraging the aspect that single-stranded RNA as compared todouble-stranded DNA, is generally more accessible to oligonucleotidemediated targeting without a need for additional enabling proteins, andbuilding on the advances in tRNA mediated codon suppression and geneticcode expansion, as well as adenosine deaminase mediated RNA editing,Applicants have engineered and optimized an integrated platform for RNAtargeting, and demonstrate its efficacy in in vitro and in vivoapplications.

Vector Design and Construction

To construct the GFP reporters—GFP-Amber, GFP-Ochre and GFP-Opal, threegene blocks were synthesized with ‘TAG’, ‘TAA’ and ‘TGA’ respectivelyreplacing the Y39 residue of the wild type GFP and were cloneddownstream of a CAG promoter. One, two, or four copies of the endogenoussuppressor tRNAs were cloned into an AAV vector containing a human U6and mouse U6 promoter. Pyrrolysyl tRNAs and adRNAs/radRNAs weresimilarly cloned into an AAV vector containing a human U6 and mouse U6promoter along with a CMV promoter driving the expression ofMbPylRS/MmPylRS/AcKRS or hADAR2 respectively.

Mammalian Cell Culture and Transfection

All HEK 293T cells were grown in Dulbecco's Modified Eagle Mediumsupplemented with 10% FBS and 1% Antibiotic-Antimycotic (Thermo Fisher)in an incubator at 37° C. and 5% CO₂ atmosphere. All in vitrotransfection experiments were carried out in HEK 293T cells using thecommercial transfection reagent Lipofectamine 2000 (Thermo Fisher). Allin vitro suppression and editing experiments were carried out in 24 wellplates using 500 ng of reporter plasmid and 1000 ng of the suppressortRNA/aaRS plasmid or the adRNA/ADAR2 plasmid. Cells were transfected at30% confluence. Cells were harvested 48 and 72 hours post transfectionfor quantification of suppression and editing respectively. The UAAsNε-Boc-L-Lysine (Chemimpex) and Nε-Acetyl-L-Lysine (Sigma) were added tothe media at the desired concentration before transfection.

Production of AAV Vectors

Virus was prepared using the protocol from the Gene Transfer, Targetingand Therapeutics (GT3) core at the Salk Institute of Biological Studies(La Jolla, Calif.). AAV8 particles were produced using HEK 293T cellsvia the triple transfection method and purified via an iodixanolgradient. Confluency at transfection was about 80%. Two hours prior totransfection, DMEM supplemented with 10% FBS was added to the HEK 293Tcells. Each virus was produced in 5×15 cm plates, where each plate wastransfected with 7.5 ug of pXR-8, 7.5 of ug recombinant transfer vector,7.5 ug of pHelper vector using PEI (1 ug/uL linear PEI in 1×DPBS pH 4.5,using HCl) at a PEI:DNA mass ratio of 4:1. The mixture was incubated for10 minutes at RT and then applied dropwise onto the cell media. Thevirus was harvested after 72 hours and purified using an iodixanoldensity gradient ultracentrifugation method. The virus was then dialyzedwith 1×PBS (pH 7.2) supplemented with 50 mM NaCl and 0.0001% of PluronicF68 (Thermo Fisher) using 50 kDA filters (Millipore), to a final volumeof ˜1 mL and quantified by qPCR using primers specific to the ITRregion, against a standard (ATCC VR-1616).

AAV-ITR-F: 5 ‘-CGGCCTCAGTGAGCGA-3’ (SEQ ID NO: 158) and

AAV-ITR-R: 5 ‘-GGAACCCCTAGTGATGGAGTT-3’ (SEQ ID NO: 159).

RNA Isolation and Next Generation Sequencing Library Preparation

RNA from gastrocnemius or TA muscles of mdx mice or livers of spf^(ash)mice was extracted using the RNeasy Plus Universal Mini Kit (Qiagen),according to the manufacturer's protocol. Next generation sequencinglibraries were prepared as follows. cDNA was synthesized using theProtoscript II First Strand cDNA synthesis Kit (New England Biolabs).Briefly, 500 ng of input cDNA was amplified by PCR with primers thatamplify 150 bp surrounding the sites of interest using KAPA HifiHotStart PCR Mix (Kapa Biosystems). PCR products were gel purified(Qiagen Gel Extraction kit), and further purified (Qiagen PCRPurification Kit) to eliminate byproducts. Library construction was donewith NEBNext Multiplex Oligos for Illumina kit (NEB). 10 ng of input DNAwas amplified with indexing primers. Samples were then pooled and loadedon an Illumina Miseq (150 single-end run) at 5 nM concentrations. Dataanalysis was performed using CRISPResso.

Animal Experiments

AAV Injections: All animal procedures were performed in accordance withprotocols approved by the Institutional Animal Care and Use Committee(IACUC) of the University of California, San Diego. All mice wereacquired from Jackson labs. AAVs were injected into the gastrocnemius orTA muscle of mdx mice (6-10 weeks old) using 2.5E+12 vg/muscle. AAVswere injected into spf^(ash) (10-12 weeks old) mice via retro-orbitalinjections using 3E+12 vg/mouse.

UAA Administration: Mice were fed water containing 20 mg/mlNε-Boc-L-Lysine (Chemimpex) for one month. Mice were also administeredthe 30 mg Nε-Boc-L-Lysine via IP injections, thrice a week.

Immunofluorescence

Harvested gastrocnemius or TA muscles were placed in molds containingOCT compound (VWR) and flash frozen in liquid nitrogen. 20 μm sectionswere cut onto pre-treated histological slides. Slides were fixed using4% Paraformaldehyde. Dystrophin was detected with a rabbit polyclonalantibody against the N-terminal domain of dystrophin (1:100, Abcam15277) followed by a donkey anti-rabbit Alexa 546 secondary antibody(1:250, Thermo Fisher).

Statistical Analysis

All statistical analyses were performed using the software GraphpadPrism and p-values were computed by unpaired two-tailed t tests.

Results

Applicants focused first on establishing the system for targetingnonsense mutations. This was motivated by the fact that nonsensemutations are responsible for 11% of all described gene lesions causinginheritable human disease, and close to 20% of disease-associated singlebase substitutions that affect the coding regions of genes.Specifically, we explored two independent but complementary approachesto directly target nonsense mutations. First, Applicants focused onengineering robust nonsense codon suppression via suppressor tRNAs.Although the use of suppressor tRNAs for premature stop codonread-through of endogenous non-sense mutations has been attempted invivo in mice, these prior studies relied only on plasmid delivery andthe use of robust and optimized delivery formats was not explored.Additionally, the potential use of un-natural amino acid (UAA) basedinducible in vivo suppression of a disease-causing endogenous nonsensemutation has not been explored either. Towards this, Applicants firstmodified the anticodon stems of serine, arginine and leucine tRNAs tocreate suppressor tRNAs targeting all three stop codons, amber, opal andochre, and evaluated these constructs in cells using GFP reportersharboring corresponding nonsense mutations. Among these, the serinesuppressor tRNA demonstrated the most consistent and robust results(FIG. 16A, FIG. 18A). To also engineer UAA mediated inducible codonsuppression, we next utilized the pyrrolysyl-tRNA/aminoacyl tRNAsynthetase (aaRS) pair from Methanosarcina barkeri (MbPylRS)^(32,33) andcloned it into AAV vectors. This enabled programmable incorporation ofUAAs at a stop codon. Notably, Applicants found that adding a secondcopy of the tRNA into the expression vector significantly boostedsuppression efficiencies (FIG. 18B). Applicants further systematicallyevaluated additional aminoacyl tRNA synthetases from Methanosarcinamazei (MmPylRS)³⁴ and an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), andalso explored varying the number of tRNAs copies per vector to up tofour (FIG. 18B).

As suppressor tRNA based approaches can lead to the read-through ofother non-target stop codons, concurrently Applicants also engineered asystem for sequence-specific targeted RNA editing via adenosinedeaminase enzymes. Specifically, adenosine to inosine (A to I) editingis a common post-transcriptional modification in RNA, catalyzed byadenosine deaminases acting on RNA (ADARs). Inosine is a deaminated formof adenosine and is biochemically recognized as guanine. Recently,multiple studies have demonstrated the engineering of ADAR2 mediatedtargeting in vitro, and a study also demonstrated correction of thenonsense mutation in CFTR in xenopus oocytes. Building on this,Applicants engineered here a system for sequence-specific targeted RNAediting in vitro and in vivo, utilizing the human ADAR2 enzyme and anassociated ADAR2 guide RNA (adRNAs) engineered from its naturallyoccurring substrate GluR2 pre-mRNA. This ADAR2 guiding RNA comprises anADAR-recruiting domain and a programmable antisense region that iscomplementary to a specified target RNA sequence. Applicants firstevaluated the RNA editing efficiency of this system in vitro byco-transfecting the constructs with GFP reporters harboring a non-senseamber or ochre mutation at Y39. Specifically, Applicants utilized twoediting approaches to engineer the editing of both adenosines in theochre stop codon: a one-step mechanism where both the adenosines areedited simultaneously or a two-step mechanism wherein editing takesplace sequentially. In addition, we also explored the possibility ofconversion of an ochre codon to an amber codon followed by ambersuppression to restore GFP expression. All three approaches enabledrestoration of GFP expression (FIG. 16C, FIG. 19A). Applicants nextconstructed AAV vectors to deliver the adRNA or a reverse oriented adRNA(radRNA) along with the ADAR2 enzyme. Similar to tRNA mediated codonsuppression, addition of a second copy of the adRNA/radRNA significantlyimproved the targeting efficiency (FIG. 19D). Applicants furthersystematically evaluated modified ADAR recruiting domains, and a rangeof RNA targeting antisense designs of varying lengths and the number ofnucleotides intervening the target A and the R/G motif of the adRNA²⁶,yielding a panel of efficient adRNA designs (FIG. 19B-C).

Based on the above in vitro optimizations, Applicants next tested thesystem for in vivo RNA targeting. Applicants focused first on the mdxmouse model for Duchenne muscular dystrophy (DMD)³⁵ which bears an ochrestop site in exon 23 of the dystrophin gene. Recent studies utilizingthe CRISPR-Cas9 system have shown promising results in the prevention³⁸and partial functional recovery of DMD by making changes in exon 23 atthe DNA level in the mdx mouse. We thus concurrently evaluated threeapproaches (FIG. 17A): one, suppressor tRNAs derived from modifiedendogenous tRNAs or pyrrolysyl tRNAs for nonsense codon suppression;two, ADAR2 based correction of the nonsense mutation; and, three,CRISPR-Cas9 based genome targeting to benchmark the RNA targetingapproaches.

Corresponding, Applicants first designed an AAV carrying two copies ofthe serine suppressor tRNA targeting the ochre stop codon, and thetibalis anterior (TA) or gastrocnemius of mdx mice were injected withthe same. Mice muscles were harvested after 2, 4, and 8 weeks.Progressively improved restoration of dystrophin expression was seenover time, with the mice harvested after 8 weeks showing the greatestdegree of restoration (FIG. 17B, FIG. 20A). In addition, neuronal nitricoxide synthase (nNOS) activity was restored at the sarcolemma which isabsent in mdx mice due to the absence of the nNOS binding site in themutant dystrophin protein (FIG. 17B). To further make the systeminducible, a vector carrying two copies of the pyrrolysyl-tRNA targetingthe ochre stop codon and MbPylRS was also constructed and injected intothe TA or gastrocnemius of mdx mice, and the mice were divided into twogroups: one that was administered the pyrrolysine UAA and a controlgroup that was not. Expectedly only mice that were provided the UAAshowed nNOS localization at the sarcolemma (FIG. 20B), and restorationof dystrophin expression (FIG. 20C).

Next, Applicants evaluated the ADAR2 based site-specific RNA editingapproach in this mouse model. To test the efficiency of this system inediting both adenosines in the ochre stop codon in mdx DMD mRNA,Applicants first optimized the constructs in vitro with a reporterplasmid bearing a fragment of the mdx DMD mRNA in HEK293T cells. Sangersequencing and NGS analysis confirmed successful targeting (FIG. 21A).Applicants next packaged the optimized constructs into AAV8, andinjected the tibialis anterior (TA) or gastrocnemius of mdx mice. Eightweeks post injection, TA and gastrocnemius muscles were collected frommdx mice, wild type mice, and mice treated with adRNA targeting andnon-targeting controls. IHC revealed clear restoration of dystrophinexpression (FIG. 17B). In addition, nNOS activity was also restored atthe sarcolemma (FIG. 17B). RNA editing rates (TAA→TGG/TAG/TGA) of0.5-0.7% were observed in treated mice (FIG. 17C, FIG. 21B). Applicantsalso note that the mdx mice showed no mRNA with a TAA→TGG change whilethe treated mice showed up to 0.42% TAA→TGG edited mRNA. Applicants notethat corresponding DNA editing rates via CRISPR-Cas9 in published invivo targeting studies were about 2%³⁹. To further benchmark the tRiADapproach, we thus also targeted the mdx mice via CRISPR based genomeediting of the nonsense mutation. Applicants injected vectors bearingdual-gRNAs to excise exon 23 codon, and expectedly, this led torestoration of dystrophin expression in a subset of the muscle cells(FIG. 17B).

Finally, we also evaluated the ADAR2 mediated RNA editing approach in anindependent mouse model of human disease. Specifically, we focused onthe male sparse fur ash (spf^(ash)) mouse model of ornithinetranscarbamylase (OTC) deficiency. The spf^(ash) mice have a G→A pointmutation in the last nucleotide of the fourth exon of the OTC gene,which leads to OTC mRNA deficiency and production of mutant protein⁴³.Recent studies have demonstrated the use of CRISPR-Cas9 and homologousrecombination based strategies for robust correction of this mutation inneonatal mice. However, gene correction via homology-directed repair(HDR) in adult mice was inefficient and led to genomic deletions whichproved to be lethal as they compromised liver function in targeted mice.To test the effectiveness of the system in editing the point mutation inspf^(ash) OTC mRNA (FIG. 17D), Applicants first evaluated our constructsin vitro with a plasmid bearing a fragment of the spf^(ash) OTC mRNA inHEK293T cells. Sanger sequencing and next generation sequencing (NGS)analysis confirmed robust RNA editing efficiencies (FIG. 21C).Applicants next packaged the constructs into AAV8, which has high livertropism⁴⁴, and injected 10-12 week old spf^(ash) mice. Four weeks postinjection, Applicants collected liver samples from spf^(ash), wild-typelitter mates, and spf^(ash) mice treated with the ADAR2 targeting andnon-targeting vectors and evaluated editing efficiency via NGS. Notably,significant RNA editing rates in the range of 0.8-4.2% were observed intreated mice in the spliced OTC mRNA (FIG. 17E, FIG. 21D), furtherconfirming the utility of this approach for in vivo editing ofendogenous RNA in adult mice.

Taken together, Applicants' results establish the use of suppressortRNAs and ADAR2 as potential strategies for in vivo RNA targeting ofpoint mutations. Specifically, by optimizing delivery, Applicants firstdemonstrated robust and inducible stop codon read-through via the use ofsuppressor tRNAs. The delivery of modified endogenous suppressor tRNAsfor premature stop read-through has several potential advantages: itlacks the toxicity associated with read-through drugs such as gentamycinand can be used to bring about efficient stop codon read-through inpost-mitotic cells. In addition, being of endogenous origin, it is notlikely to elicit a strong immune response. Additionally, theinducibility enabled by the UAA based systems, albeit non-native, couldprovide tight regulation over the expression of genes. Localizedinjections of the UAA into the target muscle could further help improvethe efficiency of the system in future studies. Notably, Applicants didnot observe any overt toxicity via this approach in the mdx targetingstudies. Applicants however note too that an important caveat to thisstrategy, analogous to the read-through drugs, is that suppressor tRNAbased approaches will lead to the read-through of other non-target stopcodons. In this regard, Applicants thus also demonstrated ADAR2 basedsite-specific correction of point mutations in RNA in two independentmouse models. Applicants note that potential off-targets in RNA arelimited as compared to DNA, as the transcriptome is only a small subsetof the genome. Secondly, even if off-targets exist, the presence of an Awithin the target window is required for the enzyme to create anoff-target A→G change. Lastly, the off-target effects will be transient.Thus, overall off-target effects due to a RNA editing enzyme such asADAR2 are likely to be limited, although enzyme processivity,promiscuity, and off-target hybridization of the antisense domain of theadRNA need to be studied thoroughly. ADAR2 being of human origin is alsoless likely to elicit an immune response, while enabling moresite-specific editing of RNA compared to the suppressor tRNA approach.

Applicants also note that compared to the tRiAD based RNA targetingapproaches above, CRISPR based genome targeting approaches currentlyshow faster kinetics and greater degree of mutant protein restoration.Applicants however anticipate that systematic engineering and directedevolution of the ADAR2 could help improve the editing efficiency andalso eliminate the intrinsic biases of the ADAR2 for certain sequences,coupled with insights from studies unearthing novel regulators of ADAR2providing cues to improve its stability. In this regard, Applicantstested the ADAR2-E488Q mutant and noted that it enabled higher editingefficiency than the wild type ADAR2 for both the DMD and OTC mRNAfragments expressed in vitro (FIG. 22). The demonstration ofsite-specific A→G mRNA editing in vivo also opens up the door to futuresite-specific C→T editing via targeted recruitment of cytosinedeaminases, thereby potentially expanding the repertoire of RNA editingtools. However, an important consideration while targeting RNA for genetherapy via the use of non-integrating vectors such as AAVs, is thenecessity for periodic re-administration of the effector constructs dueto the typically limited half-life of edited mRNAs. Secondly, RNAfolding, intrinsic half-life, localization, and RNA binding proteinsmight also impact accessibility of target sites in the RNA. Forinstance, in this example, the short half-life of mutant dystrophin RNA,and the need to target the transient pre-mRNA in OTC potentiallynegatively impact overall editing efficiencies. Chemical and structuralmodifications in tRNAs and adRNAs while taking cues from the specificitystudies on sgRNAs⁴⁹, or coupling of shielding proteins, or recentlydemonstrated programmable RNA binding proteins and RNA-targetingCRISPR-Cas systems, might help improve RNA stability and specificity,and improve the efficiency of the above approaches. With progressiveimprovements, Applicants thus anticipate this integrated tRiAD toolsetwill have broad implications in both applied life sciences as well asfundamental research.

Example 6—ADAR and APOBEC Editing Efficacy

A number of ADAR scaffolds—both dual and single—were tested for efficacyin recruiting ADAR in a cell line where ADAR2 was overexpressed (FIG. 28and FIG. 29). Further assessments were made for MCP-ADAR fusionscaffolds (FIG. 30). Endogenous mRNA target editing efficiency wasassessed using scaffold v2. SEQ ID NOS 160-163 are disclosed below.

mRNA Target # # # Average RAB7A GGGAAATCCAGCTAGCGGCA 32.0% 34.1% 30.2%32.1% RAB7A GGGAAAACTGTCTAGTTCCC 28.2% 27.5% 23.0% 26.2% CCNB1TAATTGACTGGCTAGTACAG 23.8% 17.2% 21.1% 20.7% CCNB1 GAGCTTTTTGCTTAGCACTG15.1% 18.4% 17.4% 17.0%

EQUIVALENTS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this technology belongs.

The present technology illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the present technologyclaimed.

Thus, it should be understood that the materials, methods, and examplesprovided here are representative of preferred aspects, are exemplary,and are not intended as limitations on the scope of the presenttechnology.

The present technology has been described broadly and genericallyherein. Each of the narrower species and sub-generic groupings fallingwithin the generic disclosure also form part of the present technology.This includes the generic description of the present technology with aproviso or negative limitation removing any subject matter from thegenus, regardless of whether or not the excised material is specificallyrecited herein.

In addition, where features or aspects of the present technology aredescribed in terms of Markush groups, those skilled in the art willrecognize that the present technology is also thereby described in termsof any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other aspects are set forth within the following claims.

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What is claimed is:
 1. An adeno-associated viral (AAV) vector plasmidcomprising a promoter, wherein the AAV vector plasmid encodes anadenosine deaminase acting on RNA (ADAR)-based editing system comprisinga guide RNA sequence; wherein the guide RNA sequence comprises: (i) tworecruiting domains; and (ii) a targeting domain, wherein uponhybridization, the targeting domain and a target RNA form a mismatchcorresponding to a cytosine of the targeting domain and an adenosine ofthe target RNA, wherein the mismatch is centrally positioned between thetwo recruiting domains, wherein the targeting domain comprises a lengthof from 60 nucleotides to 100 nucleotides, and wherein the guide RNAsequence does not comprise a chemical modification, wherein uponadministration of the vector to a human subject, the guide RNA sequencerecruits an adenosine deaminase enzyme that is endogenous to the humansubject, and wherein the endogenous adenosine deaminase enzyme, whencontacted with the guide RNA sequence at least partially bound to thetarget RNA, performs a targeted chemical modification on the adenosineof the target RNA; and wherein the ADAR-based editing system does notcomprise an ADAR1 or ADAR2 enzyme encoded by the MV vector plasmid. 2.The AAV vector of claim 1, wherein the endogenous adenosine deaminaseenzyme is an endogenous ADAR polypeptide.
 3. The AAV vector of claim 2,wherein the endogenous ADAR polypeptide is an ADAR1 polypeptide.
 4. TheAAV vector of claim 2, wherein the endogenous ADAR polypeptide is anADAR2 polypeptide.
 5. The AAV vector of claim 1, wherein the AAV vectorplasmid is an AAV2 vector plasmid, an AAV5 vector plasmid, an AAV8vector plasmid, or an AAV9 vector plasmid.
 6. The AAV vector of claim 1,wherein the two recruiting domains each independently comprise at leastone stem-loop.
 7. The AAV vector of claim 1, wherein the targetingdomain comprises at least about 80% sequence homology to any one of SEQID NO:182-206.
 8. The AAV vector of claim 1, wherein the two recruitingdomains each independently comprise at least about 90% sequence homologyto at least about 20 contiguous nucleic acids of SEQ ID NO: 180 or 181.9. The AAV vector of claim 1, wherein one of the two recruiting domainsis proximal to the 5′ end of the targeting domain.
 10. The AAV vector ofclaim 1, wherein one of the two recruiting domains is proximal to the 3′end of the targeting domain.
 11. The AAV vector of claim 1, wherein thetarget RNA comprises a non-sense mutation or a missense mutation, andwherein the adenosine is present in the non-sense or the missensemutation.
 12. The AAV vector of claim 11, wherein the non-sense mutationor the missense mutation is implicated in a disease.
 13. The AAV vectorof claim 12, wherein the disease comprises muscular dystrophy.
 14. TheAAV vector of claim 1, wherein the target RNA does not comprise anon-sense mutation or a missense mutation.
 15. A kit comprising the AAVvector of claim 1 in a container.
 16. A method of treating a disease orcondition in a subject in need thereof comprising: administering the AAVvector of claim 1 to the subject in need thereof, wherein the disease orcondition is muscular dystrophy.
 17. The AAV vector of claim 1, whereinadministration of the vector to the human subject results in aneffective amount of the guide RNA sequence to treat a disease orcondition.
 18. The AAV vector of claim 1, wherein the targeting domaincomprises a length of about 100 nucleotides.
 19. The AAV vector of claim1, wherein the two recruiting domains each have the sequence of SEQ IDNO: 180 or 181.